vsearch

NAME
SYNOPSIS
DESCRIPTION
DELIBERATE CHANGES
NOVELTIES
EXAMPLES
AUTHORS
CITATION
REPORTING BUGS
AVAILABILITY
COPYRIGHT
SEE ALSO
VERSION HISTORY

NAME

vsearch — chimera detection, clustering, dereplication and rereplication, FASTA/FASTQ file processing, masking, pairwise alignment, searching, shuffling, sorting, subsampling, and taxonomic classification of amplicons for metagenomics, genomics, and population genetics.

SYNOPSIS

Chimera detection:

vsearch --uchime_ref fastafile (--chimeras | --nonchimeras | --uchimealns | --uchimeout) outputfile --db fastafile [options]

Clustering:

Dereplication and rereplication:

vsearch (--derep_fulllength | --derep_prefix) fastafile (--output | --uc) outputfile [options]

vsearch --rereplicate fastafile --output outputfile [options]

Extraction of sequences:

vsearch --fastx_getseq fastafile (--fastaout | --fastqout | --notmatched | --notmatchedfq) outputfile --label label [options]

vsearch --fastx_getsubseq fastafile (--fastaout | --fastqout | --notmatched | --notmatchedfq) outputfile --label label [--subseq_start position] [--subseq_end position] [options]

FASTA/FASTQ file processing:

vsearch --fastq_chars fastqfile [options]

vsearch --fastq_convert fastqfile --fastqout outputfile [options]

vsearch (--fastq_eestats | --fastq_eestats2) fastqfile --output outputfile [options]

vsearch --fastq_join fastqfile --reverse fastqfile (--fastaout | --fastqout) outputfile [options]

vsearch --fastq_stats fastqfile [--log logfile] [options]

vsearch --fastx_revcomp inputfile (--fastaout | --fastqout) outputfile [options]

vsearch --sff_convert sff-file --fastqout outputfile [options]

Masking:

vsearch --fastx_mask fastxfile (--fastaout | --fastqout) outputfile [options]

vsearch --maskfasta fastafile --output outputfile [options]

Pairwise alignment:

Restriction site cutting:

vsearch --cut fastafile --cut_pattern pattern (--fastaout | --fastaout_rev | --fastaout_discarded | --fastaout_discarded_rev) outputfile [options]

Searching:

Shuffling and sorting:

vsearch (--shuffle | --sortbylength | --sortbysize) fastafile --output outputfile [options]

Subsampling:

vsearch --fastx_subsample fastafile (--fastaout | --fastqout) outputfile (--sample_pct real | --sample_size positive integer) [options]

Taxonomic classification:

vsearch --sintax fastafile --db fastafile --tabbedout outputfile [--sintax_cutoff real] [options]

UDB database handling:

vsearch --makeudb_usearch fastafile --output outputfile [options]

vsearch --udb2fasta udbfile --output outputfile [options]

vsearch (--udbinfo | --udbstats) udbfile [options]

DESCRIPTION

Environmental or clinical molecular diversity studies generate large volumes of amplicons (e.g.; SSU-rRNA sequences) that need to be checked for chimeras, dereplicated, masked, sorted, searched, clustered or compared to reference sequences. The aim of vsearch is to offer a all-in-one open source tool to perform these tasks, using optimized algorithm implementations and harvesting the full potential of modern computers, thus providing fast and accurate data processing.

Comparing nucleotide sequences is at the core of vsearch. To speed up comparisons, vsearch implements an extremely fast Needleman-Wunsch algorithm, making use of the Streaming SIMD Extensions (SSE2) of post-2003 x86-64 CPUs. If SSE2 instructions are not available, vsearch exits with an error message. On Power8 CPUs it will use AltiVec/VSX/VMX instructions. Memory usage increases rapidly with sequence length: for example comparing two sequences of length 1 kb requires 8 MB of memory per thread, and comparing two 10 kb sequences requires 800 MB of memory per thread. For comparisons involving sequences with a length product greater than 25 million (for example two sequences of length 5 kb), vsearch uses a slower alignment method described by Hirschberg (1975) and Myers and Miller (1988), with much smaller memory requirements.

Input

vsearch accept as input fasta or fastq files containing one or several nucleotidic entries. In fasta files, each nucleotidic entry is made of a header and a sequence. The header is defined as the string comprised between the ’>’ symbol and the first space, tab or the end of the line, whichever comes first. Additionally, if the header matches integer as the number of occurrences (or abundance) of the sequence in the study. That abundance information is used or created during chimera detection, clustering, dereplication, sorting and searching.

The sequence is defined as a string of IUPAC symbols (ACGTURYSWKMDBHVN), starting after the end of the identifier line and ending before the next identifier line, or the file end. vsearch silently ignores ascii characters 9 to 13, and exits with an error message if ascii characters 0 to 8, 14 to 31, ’.’ or ’-’ are present. All other ascii or non-ascii characters are stripped and complained about in a warning message.

In fastq files, each entry is made of sequence header starting with a symbol ’@’, a nucleotidic sequence (same rules as for fasta sequences), a quality header starting with a symbol ’+’ and a string of ASCII characters (offset 33 or 64), each one encoding the quality value of the corresponding position in the nucleotidic sequence.

vsearch operations are case insensitive, except when soft masking is activated. Masking is automatically applied during chimera detection, clustering, masking, pairwise alignment and searching. Soft masking is specified with the options ’--dbmask soft’ (for searching and chimera detection with a reference) or ’--qmask soft’ (for searching, de novo chimera detection, clustering and masking). When using soft masking, lower case letters indicate masked symbols, while upper case letters indicate regular symbols. Masked symbols are never included in the unique index words used for sequence comparisons, otherwise they are treated as normal symbols.

When comparing sequences during chimera detection, dereplication, searching and clustering, T and U are considered identical, regardless of their case. When aligning sequences, identical symbols will receive a positive match score (default +2). If two symbols are not identical, their alignment result in a negative mismatch score (default -4). Aligning a pair of symbols where at least one of them is an ambiguous symbol (BDHKMNRSVWY) will always result in a score of zero. Alignment of two identical ambiguous symbols (for example, R vs R) also receives a score of zero. When computing the amount of similarity by counting matches and mismatches after alignment, ambiguous nucleotide symbols will count as matching to other symbols if they have at least one of the nucleotides (ACGTU) they may represent in common. For example: W will match A and T, but also any of MRVHDN. When showing alignments (for example with the --alnout option) matches involving ambiguous symbols will be shown with a plus character (+) between them while exact matches between non-ambiguous symbols will be shown with a vertical bar character (|).

vsearch can read data from standard files and write to standard files, but it can also read from pipes and write to pipes! For example, multiple fasta files can be piped into vsearch for dereplication. To do so, file names can be replaced with:

	-	the symbol ’-’, representing ’/dev/stdin’ for input files or ’/dev/stdout’ for output files,
	-	a named pipe created with the command mkfifo,
	-	a process substitution ’<(command)’ as input or ’>(command)’ as output.

vsearch can automatically read compressed gzip or bzip2 files if the appropriate libraries are present during the compilation. vsearch can also read pipes streaming compressed gzip or bzip2 data if the options --gzip_decompress or --bzip2_decompress are selected. When reading from a pipe, the progress indicator is not updated.

Options

vsearch recognizes a large number of command-line commands and options. For easier navigation, options are grouped below by theme (chimera detection, clustering, dereplication and rereplication, FASTA/FASTQ file processing, masking, pairwise alignment, searching, shuffling, sorting, and subsampling). We start with the general options that apply to all themes. Options start with a double dash (--). A single dash (-) may also be used, except on NetBSD systems. Option names may be shortened as long as they are not ambiguous (e.g. --derep_f).

Help and version commands:

--help --h

Display help text with brief information about all commands and options.

--version --v

Output version information and a citation for the VSEARCH publication. Show the status of the support for gzip- and bzip2-compressed input files.

General options:

--bzip2_decompress

When reading from a pipe streaming bzip2-compressed data, decompress the data. That option is not needed when reading from a standard bzip2-compressed file.

--fasta_width positive integer

Fasta files produced by vsearch are wrapped (sequences are written on lines of integer nucleotides, 80 by default). Set that value to zero to eliminate the wrapping.

--gzip_decompress

When reading from a pipe streaming gzip-compressed data, decompress the data. That option is not needed when reading from a standard gzip-compressed file.

--log filename

Write messages to the specified log file. Information written includes program version, amount of memory available, number of cores and command line options, and if need be, informational messages, warnings and fatal errors. The start and finish times are also recorded as well as the elapsed time and the maximum amount of memory consumed. The different vsearch commands can also write additional informations to the log file.

--maxseqlength positive integer

All vsearch operations discard sequences of length equal or greater than integer (50,000 nucleotides by default).

--minseqlength positive integer

All vsearch operations discard sequences of length smaller than integer: 1 nucleotide by default for sorting or shuffling, 32 nucleotides for clustering, dereplication or searching.

--no_progress

Do not show the gradually increasing progress indicator.

--notrunclabels

Do not truncate sequence labels at first space or tab, use the full header in output files.

--quiet

Suppress all messages to stdout and stderr except for warnings and fatal error messages.

--threads positive integer

Number of computation threads to use (1 to 1024). The number of threads should be lesser or equal to the number of available CPU cores. The default is to use all available resources and to launch one thread per logical core. The following commands are multi-threaded: allpairs_global, cluster_fast, cluster_size, cluster_smallmem, cluster_unoise, fastq_mergepairs, fastx_mask, maskfasta, search_exact, sintax, uchime_ref, and usearch_global. Only one thread is used for the other commands.

Chimera detection options:

Chimera detection is based on a scoring function controlled by five options (--dn, --mindiffs, --mindiv, --minh, --xn). Sequences are first sorted by decreasing abundance, if available, and compared on their plus strand only (case insensitive).

Input sequences are masked as specified with the --qmask and --hardmask options. Masking of the database for reference based chimera detection is specified with the --dbmask option.

In de novo mode, input fasta file should present abundance annotations (i.e. a pattern [;]size=integer[;] in the fasta header). Input order matters for chimera detection, so we recommend to sort sequences by decreasing abundance (default of --derep_fulllength command). If your sequence set needs to be sorted, please see the --sortbysize command in the sorting section.

--abskew real

When using --uchime_denovo, the abundance skew is used to distinguish in a three-way alignment which sequence is the chimera and which are the parents. The assumption is that chimeras appear later in the PCR amplification process and are therefore less abundant than their parents. For --uchime3_denovo the default value is 16.0. For the other commands, the default value is 2.0, which means that the parents should be at least 2 times more abundant than their chimera. Any positive value equal or greater than 1.0 can be used.

--alignwidth positive integer

When using --uchimealns, set the width of the three-way alignments (80 nucleotides by default). Set to zero to eliminate wrapping.

--borderline filename

Output borderline chimeric sequences to filename, in fasta format. Borderline chimeric sequences are sequences that have a high enough score but which are not sufficiently different from their closest parent.

--chimeras filename

Output chimeric sequences to filename, in fasta format. Output order may vary when using multiple threads.

--db filename

When using --uchime_ref, detect chimeras using the fasta-formatted reference sequences contained in filename. Reference sequences are assumed to be chimera-free. Chimeras cannot be detected if their parents, or sufficiently close relatives, are not present in the database.

--dn real

No vote pseudo-count, corresponding to the parameter n in the chimera scoring function (default value is 1.4).

--fasta_score

Add the chimera score to the headers in the fasta output files for chimeras, non-chimeras and borderline sequences, using the format

--mindiffs positive integer

Minimum number of differences per segment (default value is 3). The parameter is ignored with --uchime2_denovo and --uchime3_denovo.

--mindiv real

Minimum divergence from closest parent (default value is 0.8). The parameter is ignored with --uchime2_denovo and --uchime3_denovo.

--minh real

Minimum score (h). Increasing this value tends to reduce the number of false positives and to decrease sensitivity. Default value is 0.28, and values ranging from 0.0 to 1.0 included are accepted. The parameter is ignored with --uchime2_denovo and --uchime3_denovo.

--nonchimeras filename

Output non-chimeric sequences to filename, in fasta format. Output order may vary when using multiple threads.

--relabel string

Relabel sequences using the prefix string and a ticker (1, 2, 3, etc.) to construct the new headers. Use --sizeout to conserve the abundance annotations.

--relabel_keep

When relabelling, keep the old identifier in the header after a space.

--relabel_md5

Relabel sequences using the MD5 message digest algorithm applied to each sequence. Former sequence headers are discarded. The sequence is converted to upper case and each ’U’ is replaced by a ’T’ before computation of the digest. The MD5 digest is a cryptographic hash function designed to minimize the probability that two different inputs give the same output, even for very similar, but non-identical inputs. Still, there is a very small, but non-zero, probability that two different inputs give the same digest (i.e. a collision). MD5 generates a 128-bit (16-byte) digest that is represented by 16 hexadecimal numbers (using 32 symbols among 0123456789abcdef). Use --sizeout to conserve the abundance annotations.

--relabel_sha1

Relabel sequences using the SHA1 message digest algorithm applied to each sequence. It is similar to the --relabel_md5 option but uses the SHA1 algorithm instead of the MD5 algorithm. SHA1 generates a 160-bit (20-byte) digest that is represented by 20 hexadecimal numbers (40 symbols). The probability of a collision (two non-identical sequences resulting in the same digest) is smaller for the SHA1 algorithm than it is for the MD5 algorithm.

	--self	When using --uchime_ref, ignore a reference sequence when its label matches the label of the query sequence (useful to estimate false-positive rate in reference sequences).
	--selfid	When using --uchime_ref, ignore a reference sequence when its nucleotide sequence is strictly identical to the nucleotidic sequence of the query.

--sizeout

When relabelling, add abundance annotations to fasta headers (using the format ’;size=integer;’).

--uchime_denovo filename

Detect chimeras present in the fasta-formatted filename, without external references (i.e. de novo). Automatically sort the sequences in filename by decreasing abundance beforehand (see the sorting section for details). Multithreading is not supported.

--uchime2_denovo filename

Detect chimeras present in the fasta-formatted filename, using the UCHIME2 algorithm. This algorithm is designed for denoised amplicons (see --cluster_unoise). Automatically sort the sequences in filename by decreasing abundance beforehand (see the sorting section for details). Multithreading is not supported.

--uchime3_denovo filename

Detect chimeras present in the fasta-formatted filename, using the UCHIME2 algorithm. The only difference from --uchime2_denovo is that the default minimum abundance skew (--abskew) is set to 16.0 rather than 2.0.

--uchime_ref filename

Detect chimeras present in the fasta-formatted filename by comparing them with reference sequences (option --db). Multithreading is supported.

--uchimealns filename

Write the three-way global alignments (parentA, parentB, chimera) to filename using a human-readable format. Use --alignwidth to modify alignment length. Output order may vary when using multiple threads. All sequences are converted to upper case before alignment. Lower case letters indicate disagreement in the alignment.

--uchimeout filename

Write chimera detection results to filename using a 18-field, tab-separated uchime-like format. Use --uchimeout5 to use a format compatible with usearch v5 and earlier versions. Rows output order may vary when using multiple threads.

	1.	score: higher score means a more likely chimeric alignment.
	2.	Q: query sequence label.
	3.	A: parent A sequence label.
	4.	B: parent B sequence label.
	5.	T: top parent sequence label (i.e. parent most similar to the query). That field is removed when using --uchimeout5.
	6.	idQM: percentage of similarity of query (Q) and model (M) constructed as a part of parent A and a part of parent B.
	7.	idQA: percentage of similarity of query (Q) and parent A.
	8.	idQB: percentage of similarity of query (Q) and parent B.
	9.	idAB: percentage of similarity of parent A and parent B.
	10.	idQT: percentage of similarity of query (Q) and top parent (T).
	11.	LY: yes votes in the left part of the model.
	12.	LN: no votes in the left part of the model.
	13.	LA: abstain votes in the left part of the model.
	14.	RY: yes votes in the right part of the model.
	15.	RN: no votes in the right part of the model.
	16.	RA: abstain votes in the right part of the model.
	17.	div: divergence, defined as (idQM - idQT).
	18.	YN: query is chimeric (Y), or not (N), or is a borderline case (?).

--uchimeout5

When using --uchimeout, write chimera detection results using a 17-field, tab-separated uchime-like format (drop the 5th field of --uchimeout), compatible with usearch version 5 and earlier versions.

--xn real

No vote weight, corresponding to the parameter beta in the scoring function (default value is 8.0).

--xsize

Strip abundance information from the headers when writing the output file.

Clustering options:

vsearch implements a single-pass, greedy centroid-based clustering algorithm, similar to the algorithms implemented in usearch, DNAclust and sumaclust for example. Important parameters are the global clustering threshold (--id) and the pairwise identity definition (--iddef).

Input sequences are masked as specified with the --qmask and --hardmask options.

--biomout filename

Generate an OTU table in the biom version 1.0 JSON file format as specified at (link) <http://biom-format.org/documentation/format_versions/biom-1.0.html>. The format describes how to store a sparse matrix containing the abundances of the OTUs in the different samples. This format is much more efficient than the classic and mothur OTU table formats available with the --otutabout and --mothur_shared_out options, respectively, and is recommended at least for large tables. The OTUs are represented by the cluster centroids. Taxonomy information will be included for the OTUs if available. Sample identifiers will be extracted from the headers of all sequences in the input file. If the header contains ’;sample=abc123;’ or ’;barcodelabel=abc123;’ or a similar string somewhere, then the given sample identifier (here ’abc123’) will be used. The semicolon is not mandatory at the beginning or end of the header. The sample identifier may contain any printable character except semicolons. If no such sample label is found, the identifier in the initial part of the header will be used, but only letters, digits and underscores are allowed. OTU identifiers will be extracted from the headers of the cluster centroid sequences. If the header contains ’;otu=def789;’ or a similar string somewhere, then the given OTU identifier (here ’def789’) will be used. The semicolon is not mandatory at the beginning or end of the header. The OTU identifier may contain any printable character except semicolons. If no such OTU label is found, the identifier in the initial part of the header will be used, and all characters except semicolons are allowed. Alternatively, OTU identifers can be generated using the relabelling options (--relabel, --relabel_sha1 or --relabel_md5). Taxonomy information, if present, will also be extracted from the headers of the centroid sequences. If the header contains ’;tax=Homo_sapiens;’ or a similar string somewhere, then the given taxonomy information (here ’Homo_sapiens’) will be used. The semicolon is not mandatory at the beginning or end of the header. The taxonomy information may contain any printable character except semicolons. If an OTU table in the biom version 2.1 HDF5 file format is required, the biom utility may be used as described at (link) <http://biom-format.org/documentation/biom_conversion.html>.

--centroids filename

Output cluster centroid sequences to filename, in fasta format. The centroid is the sequence that seeded the cluster (i.e. the first sequence of the cluster).

--clusterout_id

Add cluster identifier information to the output files when using the --consout and --profile options.

--clusterout_sort

Sort output files by decreasing abundance when using the --consout, --msaout and --profile options.

--cluster_fast filename

Clusterize the fasta sequences in filename, automatically sort by decreasing sequence length beforehand.

--cluster_size filename

Clusterize the fasta sequences in filename, automatically sort by decreasing sequence abundance beforehand.

--cluster_smallmem filename

Clusterize the fasta sequences in filename without automatically modifying their order beforehand. Sequence are expected to be sorted by decreasing sequence length, unless --usersort is used.

--cluster_unoise filename

Perform denoising of the fasta sequences in filename according to the UNOISE version 3 algorithm by Robert Edgar, but without the chimera removal step. The options --minsize (default 8) and --unoise_alpha (default 2.0) may be specified. Chimera removal (de novo) should be performed afterwards with --uchime3_denovo.

--clusters string

Output each cluster to a separate fasta file using the prefix string and a ticker (0, 1, 2, etc.) to construct the path and filenames.

--consout filename

Output cluster consensus sequences to filename. For each cluster, a multiple alignment is computed, and a consensus sequence is constructed by taking the majority symbol (nucleotide or gap) from each column of the alignment. Columns containing a majority of gaps are skipped, except for terminal gaps. If the --sizein option is specified, sequence abundances will be taken into account.

--cons_truncate

This command is ignored. A warning is issued.

--id real

Do not add the target to the cluster if the pairwise identity with the centroid is lower than real (value ranging from 0.0 to 1.0 included). The pairwise identity is defined as the number of (matching columns) / (alignment length - terminal gaps). That definition can be modified by --iddef.

--iddef 0|1|2|3|4

Change the pairwise identity definition used in --id. Values accepted are:

	0.	CD-HIT definition: (matching columns) / (shortest sequence length).
	1.	edit distance: (matching columns) / (alignment length).
	2.	edit distance excluding terminal gaps (same as --id).
	3.	Marine Biological Lab definition counting each gap opening (internal or terminal) as a single mismatch, whether or not the gap was extended: 1.0 - [(mismatches + gap openings)/(longest sequence length)]
	4.	BLAST definition, equivalent to --iddef 1 in a context of global pairwise alignment.

--minsize positive integer

Specify the minimum abundance of sequences for denoising using --cluster_unoise. The default is 8.

--msaout filename

Output a multiple sequence alignment and a consensus sequence for each cluster to filename, in fasta format. Be warned that vsearch computes center star multiple sequence alignments using a fast method whose accuracy can decrease significantly when using low pairwise identity thresholds. The consensus sequence is constructed by taking the majority symbol (nucleotide or gap) from each column of the alignment. Columns containing a majority of gaps are skipped, except for terminal gaps. If the --sizein option is specified, sequence abundances will be taken into account when computing the consensus.

--mothur_shared_out filename

Output an OTU table in the mothur ’shared’ tab-separated plain text format as described at (link) <https://www.mothur.org/wiki/Shared_file>. The format describes how a matrix containing the abundances of the OTUs in the different samples is stored. The first line will start with the strings ’label’, ’group’ and ’numOtus’ and is followed by a list of all OTU identifiers. The following lines, one for each sample, starts with the string ’vsearch’ followed by the sample identifier, the total number of OTUs, and a list of abundances for each OTU in that sample, in the order given on the first line. The OTU and sample identifiers are extracted from the FASTA headers of the sequences. The OTUs are represented by the cluster centroids. See the --biomout option for further details.

--otutabout filename

Output an OTU table in the classic tab-separated plain text format as a matrix containing the abundances of the OTUs in the different samples. The first line will start with the string ’#OTU ID’ and is followed by a tab-separated list of all sample identifiers. The following lines, one for each OTU, starts with the OTU identifier and is followed by a tab-separated list of abundances for that OTU in each sample, in the order given on the first line. The OTU and sample identifiers are extracted from the FASTA headers of the sequences. The OTUs are represented by the cluster centroids. An extra column is added to the right of the table if taxonomy information is available for at least one of the OTUs. This column will be labelled ’taxonomy’ and each row will then contain the taxonomy information extracted for that OTU. See the --biomout option for further details.

--profile filename

Output a sequence profile to a text file with the frequency of each nucleotide in each position in the multiple alignment for each cluster. There is a FASTA-like header line for each cluster, followed by the profile information in a tab-separated format. The eight columns are: position (0-based), consensus nucleotide, number of As, number of Cs, number of Gs, number of Ts or Us, number of gap symbols, and finally the total number of ambiguous nucleotide symbols (B, D, H, K, M, N, R, S, Y, V or W). All numbers are integers. If the --sizein option is specified, sequence abundances will be taken into account.

--qmask none|dust|soft

Mask regions in sequences using the dust or the soft methods, or do not mask (none). Warning, when using soft masking, clustering becomes case sensitive. The default is to mask using dust.

--relabel string

Relabel sequence identifiers in the output files produced by --consout, --profile and --centroids options. Please see the description of the same option under Chimera detection for details.

--relabel_keep

When relabelling, keep the old identifier in the header after a space.

--relabel_md5

Relabel sequence identifiers in the output files produced by --consout, --profile and --centroids options. Please see the description of the same option under Chimera detection for details.

--relabel_sha1

Relabel sequence identifiers in the output files produced by --consout, --profile and --centroids options. Please see the description of the same option under Chimera detection for details.

--sizein

Take into account the abundance annotations present in the input fasta file (search for the pattern ’[>;]size=integer[;]’ in sequence headers).

--sizeorder

When an amplicon is close to 2 or more centroids, both within the distance specified with the --id option, resolve the ambiguity by clustering it with the centroid having the highest abundance, not necessarily the closest one. The option only has effect when the value specified with --maxaccepts is higher than one. The --sizeorder option turns on what is sometimes referred to as abundance-based greedy clustering (AGC), in contrast to the default distance-based greedy clustering (DGC).

--sizeout

Add abundance annotations to the output fasta files (add the pattern specified, abundance annotations are reported to output files, and each cluster centroid receives a new abundance value corresponding to the total abundance of the amplicons included in the cluster (--centroids option). If --sizein is not specified, input abundances are set to 1 for amplicons, and to the number of amplicons per cluster for centroids.

--strand plus|both

When comparing sequences with the cluster seed, check the plus strand only (default) or check both strands.

--uc filename

Output clustering results in filename using a tab-separated uclust-like format with 10 columns and 3 different type of entries (S, H or C). Each fasta sequence in the input file can be either a cluster centroid (S) or a hit (H) assigned to a cluster. Cluster records (C) summarize information (size, centroid label) for each cluster. In the context of clustering, the option --uc_allhits has no effect on the --uc output. Column content varies with the type of entry (S, H or C):

	1.		Record type: S, H, or C.
	2.		Cluster number (zero-based).
	3.		Centroid length (S), query length (H), or cluster size (C).
	4.		Percentage of similarity with the centroid sequence (H), or set to ’*’ (S, C).
	5.		Match orientation + or - (H), or set to ’*’ (S, C).
	6.		Not used, always set to ’*’ (S, C) or to zero (H).
	7.		Not used, always set to ’*’ (S, C) or to zero (H).
	8.		set to ’*’ (S, C) or, for H, compact representation of the pairwise alignment using the CIGAR format (Compact Idiosyncratic Gapped Alignment Report): M (match/mismatch), D (deletion) and I (insertion). The equal sign ’=’ indicates that the query is identical to the centroid sequence.
	9.		Label of the query sequence (H), or of the centroid sequence (S, C).
	10.		Label of the centroid sequence (H), or set to ’*’ (S, C).

--unoise_alpha real

Specify the alpha parameter to the --cluster_unoise command. The default i 2.0.

--usersort

When using --cluster_smallmem, allow any sequence input order, not just a decreasing length ordering.

	--xsize		Strip abundance information from the headers when writing the output file.
	...		Most searching options as well as score filtering, gap penalties and masking also apply to clustering (see the Searching section for definitions): --alnout, --blast6out, --fastapairs, --matched, --notmatched, --maxaccept, --maxreject, --samout, --userout, --userfields

Dereplication and rereplication options:

--derep_fulllength filename

Merge strictly identical sequences contained in filename. Identical sequences are defined as having the same length and the same string of nucleotides (case insensitive, T and U are considered the same). See the options --sizein and --sizeout to take into account and compute abundance values. This command does not support multithreading.

--derep_prefix filename

Merge sequences with identical prefixes contained in filename. A short sequence identical to an initial segment (prefix) of another sequence is considered a replicate of the longer sequence. If a sequence is identical to the prefix of two or more longer sequences, it is clustered with the shortest of them. If they are equally long, it is clustered with the most abundant. Remaining ties are solved using sequence headers and sequence input order. Sequence comparisons are case insensitive, and T and U are considered identical. This command does not support multithreading.

--maxuniquesize positive integer

Discard sequences with a post-dereplication abundance value greater than integer.

--minuniquesize positive integer

Discard sequences with a post-dereplication abundance value smaller than integer.

--output filename

Write the dereplicated sequences to filename, in fasta format and sorted by decreasing abundance. Identical sequences receive the header of the first sequence of their group. If --sizeout is used, the number of occurrences (i.e. abundance) of each sequence is indicated at the end of their fasta header using the pattern

			--relabel string Please see the description of the same option under Chimera detection for details.
			--relabel_keep When relabelling, keep the old identifier in the header after a space.
			--relabel_md5 Please see the description of the same option under Chimera detection for details.
			--relabel_sha1 Please see the description of the same option under Chimera detection for details.
			--rereplicate filename Duplicate each sequence the number of times indicated by the abundance of each sequence in the specified file (option --sizein is always implied). The sequence labels are identical for the same sequence, unless --relabel, --relabel_sha1 or --relabel_md5 is used to create unique labels. Output is written to the file specified with the --output option, in FASTA format. The output file does not contain abundance information unless --sizeout is specified, in which case an abundance of 1 is used.
	--sizein		Take into account the abundance annotations present in the input fasta file (search for the pattern ’[>;]size=integer[;]’ in sequence headers). That option is active by default when rereplicating.

--sizeout

Add abundance annotations to the output fasta file (add the pattern specified, each unique sequence receives a new abundance value corresponding to its total abundance (sum of the abundances of its occurrences). If --sizein is not specified, input abundances are set to 1, and each unique sequence receives a new abundance value corresponding to its number of occurrences in the input file.

--strand plus|both

When searching for strictly identical sequences, check the plus strand only (default) or check both strands.

--topn positive integer

Output only the top integer sequences (i.e. the most abundant).

--uc filename

Output full-length or prefix-dereplication results in filename using a tab-separated uclust-like format with 10 columns and 3 different type of entries (S, H or C). Each fasta sequence in the input file can be either a cluster centroid (S) or a hit (H) assigned to a cluster. Cluster records (C) summarize information (size, centroid label) for each cluster. In the context of dereplication, the option --uc_allhits has no effect on the --uc output. Column content varies with the type of entry (S, H or C):

	1.		Record type: S, H, or C.
	2.		Cluster number (zero-based).
	3.		Sequence length (S, H), or cluster size (C).
	4.		Percentage of similarity with the centroid sequence (H), or set to ’*’ (S, C).
	5.		Match orientation + or - (H), or set to ’*’ (S, C).
	6.		Not used, always set to ’*’ (S, C) or 0 (H).
	7.		Not used, always set to ’*’ (S, C) or 0 (H).
	8.		Not used, always set to ’*’.
	9.		Label of the query sequence (H), or of the centroid sequence (S, C).
	10.		Label of the centroid sequence (H), or set to ’*’ (S, C).

--xsize

Strip abundance information from the headers when writing the output file.

Extraction options:

Sequences with headers matching certain criteria can be extracted from FASTA and FASTQ files using the --fastx_getseq, --fastx_getseqs and --fastx_getsubseq commands.

The --fastx_getseq command requires the header to match a label specified with the --label option. If the --label_substr_match option is given, the label may be a substring located anywhere in the header, otherwise the entire header must match the label. These matches are not case-sensitive. The headers in the input file are truncated at the first space or tab character unless the --notrunclabels option is given. The matching sequences will be written to the files specified with the --fastaout and --fastqout options, in FASTA and FASTQ format, respectively. Sequences that do not match are written to the files specified with the --notmatched and --notmatchedfq options, respectively.

The --fastx_getsubseq command is similar to the --fastx_getseq command, but will extract a subsequence of the matching sequences. The start position is specifed with the --subseq_start option and the end position is specified with the --subseq_end option. The positions are 1-based, meaning that the first symbol of the sequence is at position 1. If the start or end position option is not specified, the default is to start at the first position and end at the last position in the sequence.

The --fastx_getseqs command is similar to the --fastx_getseq command but allows more flexibility in specifying the label(s) to be matched. A single label may be specified using the --label option as described above. Alternatively, a file containing a list of labels to be matched may be specified with the --labels option. The file must be a plain text file with one label on each line. The --label_word and --label_words options may be used to specify either a single word or a file containing a list of words, respectively, to be matched. Words are defined as character sequences delimited either by a character that is not alpha-numeric (A-Z, a-z, or 0-9) or by the beginning or end of the header. Word matching is case-sensitive. The --label_field option will limit the matching of words to a certain field in the header.

--fastaout filename

Write the extracted sequences in FASTA format to the file with the given name.

--fastqout filename

Write the extracted sequences in FASTQ format to the file with the given name. This option is illegal if the input is in FASTA format.

--fastx_getseq filename

Extract sequences from the given FASTA or FASTQ file. Specify a label to match using the --label option. Output files are specified with the --fastaout, --fastqout, --notmatched and --notmatchedfq options.

--fastx_getseqs filename

Extract sequences from the given FASTA or FASTQ file. Specify the label or labels to match using one of the following options: --label, --labels, --label_word, or --label_words. Output files are specified with the --fastaout, --fastqout, --notmatched and --notmatchedfq options.

--fastx_getsubseq filename

Extract a certain part of some of the sequences in the given FASTA or FASTQ file. Specify labels to match using the --label option. Specify the subsequence range to be extracted with the --subseq_start and --subseq_end options. Output files are specified with the --fastaout, --fastqout, --notmatched and --notmatchedfq options.

--label string

Specifiy the label to match in the sequence header. Unless the --label_substr_match option is given, the label must match the entire header. The comparison is not case-sensitive.

--label_field string

Specify a field name to be used when matching using the --label_word or --label_words option. The field name is a string like "abc" that must precede the word to be matched with an equals sign (=) in between. The field must be delimited by semicolons or the beginning or end of the header. The following header will match the label 123 in the field abc: "seq1;abc=123".

--label_substr_match

The labels specified with the --label or the --labels option may match anywhere in the header if this option is given. Otherwise a label needs to match the entire header.

--label_word string

Specifiy a word to match in the sequence header. Words are defined as strings delimited by either the start or end of the header or by any symbol that is not a letter (A-Z, a-z) or digit (0-9). The comparison is case-sensitive.

--label_words filename

Specify a file containing words to be matched against the sequence headers. The plain text file must contain one word on each line. Words are defined as strings delimited by either the start or end of the header or by any symbol that is not a letter (A-Z, a-z) or digit (0-9). The comparison is case-sensitive.

--labels filename

Specify a file containing labels to be matched against the sequence headers. The plain text file must contain one label on each line. Unless the --label_substr_match option is given, a label must match the entire header. The comparison is not case-sensitive.

--notmatched filename

Write the sequences that were not extracted to the file with the given name, in FASTA format.

--notmatchedfq filename

Write the sequences that were not extracted to the file with the given name, in FASTQ format. This option is illegal if the input is in FASTA format.

--subseq_end positive integer

Specifiy the end position in the sequences when extracting subsequences using the --fastx_getsubseq command. Positions are 1-based, so the sequences start at position 1. The default is to end at the end of the sequence if this option is not specified.

--subseq_start positive integer

Specifiy the starting position in the sequences when extracting subsequences using the --fastx_getsubseq command. Positions are 1-based, so the sequences start at position 1. The default is to start at the beginning of the sequence (position 1), if this option is not specified.

FASTA/FASTQ file processing options:

Analyse, trim, filter, convert or merge sequences in FASTQ files, or reverse complement sequences in FASTA or FASTQ files. The --fastq_chars command can be used to analyse FASTQ files to identify the quality encoding and the range of quality score values used. To convert between different FASTQ file variants, use the --fastq_convert command. Statistical analysis of the quality and length of the sequences in a FASTQ file may be performed with the --fastq_stats, --fastq_eestats, and --fastq_eestats2 commands. Sequences may be trimmed, filtered and converted by the --fastq_filter or --fastx_filter commands. Paired-end reads can be merged using the --fastq_mergepairs command. The --fastx_revcomp command reverse-complements sequences. Finally, the --sff_convert command can be used to convert SFF files to FASTQ.

--eeout

When using --fastq_filter or

--fastq_mergepairs, include the number of expected errors (ee) in the sequence header of FASTQ and FASTA files. This option is a synonym of the --fastq_eeout option. Use the --xee option to remove this information from headers.

--eetabbedout filename

When specified with the --fastq_mergepairs command, write statistics with expected errors of each merged read to the given file. The file is a tab separated file with four columns: The number of errors expected in the forward read, the number of expected errors in the reverse read, the number of observed errors in the forward read, and the number of observed errors in the reverse read. The observed number of errors are the number of differences in the overlap region of the merged sequence relative to each of the reads in the pair.

--fastaout filename

When using --fastq_filter, --fastq_mergepairs or --fastx_filter, write to the given FASTA-formatted file the sequences passing the filter, or the merged sequences.

--fastaout_rev filename

When using --fastq_filter, or --fastx_filter, write to the given FASTA-formatted file the reverse reads passing the filter.

--fastaout_notmerged_fwd filename

When using --fastq_mergepairs, write forward reads not merged to the specified FASTA file.

--fastaout_notmerged_rev filename

When using --fastq_mergepairs, write reverse reads not merged to the specified FASTA file.

--fastaout_discarded filename

Write sequences that do not pass the filter of the --fastq_filter or --fastx_filter command to the given FASTA-formatted file.

--fastaout_discarded_rev filename

Write reverse reads that do not pass the filter of the --fastq_filter or --fastx_filter command to the given FASTA-formatted file.

--fastq_allowmergestagger

When using --fastq_mergepairs, allow to merge staggered read pairs. Staggered pairs are pairs where the 3’ end of the reverse read has an overhang to the left of the 5’ end of the forward read. This situation can occur when a very short fragment is sequenced. The 3’ overhang of the reverse read is not included in the merged sequence. The opposite option is the --fastq_nostagger option. The default is to discard staggered pairs.

--fastq_ascii positive integer

Define the ASCII character number used as the basis for the FASTQ quality score. The default is 33, which is used by the Sanger / Illumina 1.8+ FASTQ format (phred+33). The value 64 is used by the Solexa, Illumina 1.3+ and Illumina 1.5+ formats (phred+64). Only 33 and 64 are valid arguments.

--fastq_asciiout positive integer

When using --fastq_convert or --sff_convert, define the ASCII character number used as the basis for the FASTQ quality score when writing FASTQ output files. The default is 33. Only 33 and 64 are valid arguments.

--fastq_chars filename

Summarize the composition of sequence and quality strings contained in the input FASTQ file. For each of the four DNA letters, --fastq_chars gives the number of occurrences of the letter, its relative frequency and the length of the longest run of that letter. For each character present in the quality strings, --fastq_chars gives the ASCII value of the character, its relative frequency, and the number of times a k-mer of that character appears at the end of quality strings. The length of the k-mer can be set using --fastq_tail (4 by default). The command --fastq_chars tries to automatically detect the quality encoding (Solexa, Illumina 1.3+, Illumina 1.5+ or Illumina 1.8+/Sanger) by analyzing the range of observed quality score values. In case of success, --fastq_chars suggests values for the --fastq_ascii (33 or 64), --fastq_qmin and --fastq_qmax options to be used with the other commands that require a FASTQ input file.

--fastq_convert filename

Convert between the different variants of the FASTQ file format. The quality encoding of the input file must be specified with the --fastq_ascii option (either 33 or 64, the default is 33), and the output quality encoding must be specified with the --fastq_asciiout option (default 33). The minimum and maximum output quality scores may be limited using the --fastq_qminout and --fastq_qmaxout options. The output file is specified with the --fastqout option.

--fastq_eeout

When using --fastq_filter, --fastx_filter or --fastq_mergepairs, include the number of expected errors (ee) in the sequence header of FASTQ and FASTA files. This option is a synonym of the --eeout option. Use the --xee option to remove this information from headers.

--fastq_eestats filename

Analyze a FASTQ file and report statistics on the distributions of quality scores, error probabilities and expected accumulated errors. The report, a table of 21 tab-separated columns, is written to the file specified with the --output option. The first column corresponds to the position in the reads (Pos). The second and third columns correspond to the number of reads (Reads) and percentage of reads (PctRecs) that include this position. The remaining columns include information about the distribution of quality scores in this position (Q), error probabilities in this position (Pe), and finally the expected number of accumulated errors from the beginning of the reads and until the current position (EE). For each of the Q, Pe and EE distributions, the following statistics are included: minimum value (Min), lower quartile (Low), median (Med), mean (Mean), upper quartile (Hi), and maximum value (Max). The quality encoding and the range of quality values may be specified with --fastq_ascii --fastq_qmin and --fastq_qmax.

--fastq_eestats2 filename

Analyze the specified FASTQ file and report statistics on the number of sequences that would be retained at a combination of selected cutoffs for length truncation and maximum expected errors, that could potentially be used as arguments to the --fastq_trunclen and --fastq_maxee options to the --fastq_filter command. The result, a table of two or more columns, is written to the file specified with the --output option. There is a line for each length truncation cutoff. The first column on each line contains the selected truncation length, while the following columns contain the number of sequences and, in parenthesis, the percentage of sequences that would be retained at the selected EE levels. The truncation length cutoffs may be specified with the --length_cutoffs option and requires a list of three comma-separated integers indicating the shortest cutoff, the longest cutoff, and the increment between cutoffs. The longest cutoff may be specified with a star (*) which indicates that the limit is equal to the longest sequence in the input file. The default setting is "50,*,50" meaning that truncation lengths of 50, 100, 150 and so on up to the longest sequence length should be used. The maximum expected error (EE) cutoffs may be specified with the --ee_cutoffs option which requires a comma-separated list of floating point numbers as its argument. The default setting is "0.5,1.0,2.0" that indicates that expected error levels of 0.5, 1.0 and 2.0 should be used.

--fastq_filter filename

Trim and/or filter sequences in the given FASTQ file. Similar to the --fastx_filter command, but works only on FASTQ files. See --fastx_filter for details.

--fastq_join filename

Join paired-end sequence reads into one sequence and add a gap between them using a padding sequence. The sequences are not merged as with the fastq_mergepairs command, but simply joined with a gap. The forward reads are specified as the argument to this option and the reverse reads are specified with the --reverse option. The resulting sequences consist of the forward read, the padding sequence and the reverse complement of the reverse read. The padding sequence is specified with the --join_padgap option and the padding quality is specified with the --join_padgapq option. The default padding sequence string is NNNNNNNN and the default padding quality string is IIIIIIII, corresponding to a base quality score of 40 (a very high quality score with error probability 0.0001). The joined sequences are output to the file(s) specified with the --fastaout or --fastqout options.

--fastq_maxdiffs positive integer

When using --fastq_mergepairs, specify the maximum number of non-matching nucleotides allowed in the overlap region. That option has a strong influence on the merging success rate. The default value is 10.

--fastq_maxdiffpct real

When using --fastq_mergepairs, specify the maximum percentage of non-matching nucleotides allowed in the overlap region. The default value is 100.0%. There are other more sophisticated rules in the merging algorithm that will discard read pairs with a high fraction of mismatches.

--fastq_maxee real

When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard sequences with more than the specified number of expected errors.

--fastq_maxee_rate real

When using --fastq_filter or --fastx_filter, discard sequences with more than the specified number of expected errors per base.

--fastq_maxlen positive integer

When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard sequences with more than the specified number of bases.

--fastq_maxmergelen positive integer

When using --fastq_mergepairs, specify the maximum length of the merged sequence. By default there is no limit.

--fastq_maxns positive integer

When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard sequences with more than the specified number of N’s.

--fastq_mergepairs filename

Merge paired-end sequence reads into one sequence. The forward reads are specified as the argument to this option and the reverse reads are specified with the --reverse option. The merged sequences are output to the file(s) specified with the --fastaout or --fastqout options. The non-merged reads can be output to the files specified with the --fastaout_notmerged_fwd, --fastaout_notmerged_rev, --fastqout_notmerged_fwd and --fastqout_notmerged_rev options. Statistics may be output to the file specified with the --eetabbedout option. Sequences are truncated as specified with the --fastq_truncqual option to remove low-quality bases in the 3’ end. Sequences shorter than specified with --fastq_minlen (after truncation) are discarded (1 by default). Sequences with too many ambiguous bases (N’s), as specified with the --fastq_maxns are also discarded (no limit by default). Staggered reads are not merged unless the --fastq_allowmergestagger option is specified. The minimum length of the overlap region between the reads may be specified with the --fastq_minovlen option (default 10). The overlap region may not include more mismatches than specified with the --fastq_maxdiffs option (10 by default) or a higher percentage of mismatches than specified with the --fastq_maxdiffpct option (100.0% by default), otherwise the read pair is discarded. Additional rules will avoid merging of reads that cannot be aligned reliably and unambiguously. The mimimum and maximum length of the merged sequence may be specified with the --fastq_minmergelen and --fastq_maxmergelen options, respectively. Other relevant options are: --fastq_ascii, --fastq_maxee, --fastq_nostagger, --fastq_qmax, --fastq_qmaxout, --fastq_qmin, --fastq_qminout, and --label_suffix.

--fastq_minlen positive integer

When using --fastq_filter, --fastq_mergepairs or --fastx_filter, discard sequences with less than the specified number of bases (default 1).

--fastq_minmergelen positive integer

When using --fastq_mergepairs, specify the minimum length of the merged sequence. The default is 1.

--fastq_minovlen positive integer

When using --fastq_mergepairs, specify the minimum overlap between the merged reads. The default is 10.

--fastq_nostagger

When using --fastq_mergepairs, forbid the merging of staggered read pairs. This is the default behaviour of --fastq_mergepairs. To change that behaviour, see the --fastq_allowmergestagger option.

--fastq_qmax positive integer

Specify the maximum quality score accepted when reading FASTQ files. The default is 41, which is usual for recent Sanger/Illumina 1.8+ files.

--fastq_qmaxout positive integer

When using --fastq_convert or --sff_convert, specify the maximum quality score used when writing FASTQ files. The default is 41, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may use a maximum quality score of 40.

--fastq_qmin positive integer

Specify the minimum quality score accepted for FASTQ files. The default is 0, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may use scores between -5 and 2.

--fastq_qminout positive integer

When using --fastq_convert or --sff_convert, specify the minimum quality score used when writing FASTQ files. The default is 0, which is usual for Sanger/Illumina 1.8+ files. Older versions of the format may use scores between -5 and 2.

--fastq_stats filename

Analyze a FASTQ file and report the number of reads it contains. The quality encoding and the range of quality values may be specified with --fastq_ascii --fastq_qmin and --fastq_qmax. That command requires the --log option and outputs the following detailed statistics on read length, quality score, length vs. quality distributions, and length / quality filtering:

Read length distribution:

	1.		L: read length.
	2.		N: number of reads.
	3.		Pct: fraction of reads with this length.
	4:		AccPct: fraction of reads with this length or longer.

Quality score distribution:

	1.		ASCII: character encoding the quality score.
	2.		Q: Phred quality score.
	3.		Pe: probability of error associated with the quality score.
	4.		N: number of bases with this quality score.
	5.		Pct: fraction of bases with this quality score.
	6:		AccPct: fraction of bases with this quality score or higher.

Length vs. quality distribution:

	1.		L: position in reads (starting from position 2).
	2.		PctRecs: fraction of reads with at least this length.
	3.		AvgQ: average quality score over all reads up to this position.

	4.		P(AvgQ): error probability corresponding to AvgQ.
	5.		AvgP: average error probability.
	6:		AvgEE: average expected error over all reads up to this position.
	7:		Rate: growth rate of AvgEE between this position and position - 1.
	8:		RatePct: Rate (as explained above) expressed as a percentage.

Effect of expected error and length filtering:

The first column indicates read lengths (L). The next four columns indicate the number of reads that would be retained by the --fastq_filter command if the reads were truncated at length L (option --fastq_trunclen L) and filtered to have a maximum expected error of 1.0, 0.5, 0.25 or 0.1 (with the option --fastq_maxee float). The last four columns indicate the fraction of reads that would be retained by the --fastq_filter command using the same length and maximum expected error parameters.

Effect of minimum quality and length filtering:

The first column indicates read lengths (Len). The next four columns indicate the fraction of reads that would be retained by the --fastq_filter command if the reads were truncated at length Len (option --fastq_trunclen Len) or at the first position with a quality Q below 5, 10, 15 or 20 (option --fastq_truncqual Q).

--fastq_stripleft positive integer

When using --fastq_filter or --fastx_filter, strip the specified number of bases from the left end of the reads.

--fastq_stripright positive integer

When using --fastq_filter or --fastx_filter, strip the specified number of bases from the right end of the reads.

--fastq_tail positive integer

When using --fastq_chars, count the number of times a series of characters of length k appears at the end of quality strings. By default, k = 4.

--fastq_truncee real

When using --fastq_filter or --fastx_filter, truncate sequences so that their total expected error is not higher than the specified value.

--fastq_trunclen positive integer

When using --fastq_filter or --fastx_filter, truncate sequences to the specified length. Shorter sequences are discarded.

--fastq_trunclen_keep positive integer

When using --fastq_filter or --fastx_filter, truncate sequences to the specified length. Shorter sequences are not discarded.

--fastq_truncqual positive integer

When using --fastq_filter or --fastx_filter, truncate sequences starting from the first base with the specified base quality score value or lower.

--fastqout filename

When using --fastq_filter, --fastq_mergepairs or --fastx_filter, write to the given FASTQ-formatted file the sequences passing the filter, or the merged sequences.

--fastqout_rev filename

When using --fastq_filter or --fastx_filter, write to the given FASTQ-formatted file the reverse reads passing the filter.

--fastqout_discarded filename

When using --fastq_filter or --fastx_filter, write sequences that do not pass the filter to the given FASTQ-formatted file.

--fastqout_discarded_rev filename

When using --fastq_filter or --fastx_filter, write reverse reads that do not pass the filter to the given FASTQ-formatted file.

--fastqout_notmerged_fwd filename

When using --fastq_mergepairs, write forward reads not merged to the specified FASTQ file.

--fastqout_notmerged_rev filename

When using --fastq_mergepairs, write reverse reads not merged to the specified FASTQ file.

--fastx_filter filename

Trim and/or filter the sequences in the given FASTA or FASTQ file and output the remaining sequences to the FASTQ file specified with the --fastqout option and/or to the FASTA file specified with the --fastaout option. Discarded sequences are written to the files specified with the --fastaout_discarded and --fastqout_discarded options. The input format (FASTA or FASTQ) is automatically detected. If the input consists of paired sequences, an input file with reverse reads may be specified with the --reverse option, and corresponding output will be written to the files specified with the --fastqout_rev, --fastaout_rev, --fastqout_discarded_rev, and --fastaout_discarded_rev options. Output can not be written to FASTQ files if the input is in FASTA format. The sequences are first trimmed and then filtered based on the remaining bases. Sequences may be trimmed using the options --fastq_stripleft, --fastq_stripright, --fastq_truncee, --fastq_trunclen, --fastq_trunclen_keep and --fastq_truncqual. The sequences may be filtered using the options --fastq_maxee, --fastq_maxee_rate, --fastq_maxlen, --fastq_maxns, --fastq_minlen (default 1), --fastq_trunclen, --maxsize, and --minsize. Sequences not satisfying the requirements are discarded. For pairs of sequences, both sequences in a pair must satisfy the requirements, otherwise both are discarded. If no shortening or filtering options are given, all sequences are written to the output files, possibly after conversion from FASTQ to FASTA format. The --relabel option may be used to relabel the output sequences. The --eeout option may be used to output the expected number of errors in each sequence. After all sequences have been processed, the number of kept and discarded sequences will be shown, as well as how many of the kept sequences were trimmed.

--fastx_revcomp filename

Reverse-complement the sequences in the given FASTA or FASTQ file to a file specified with the --fastaout and/or --fastqout options. If the input file is in FASTA format, the output can not be written back to a FASTQ file due to missing base quality scores.

--join_padgap string

When running --fastq_join, use the string as a sequence padding string. The default is NNNNNNNN (8 N’s).

--join_padgapq string

When running --fastq_join, use the string as a quality padding string. The default is a string of I’s equal in length to the sequence padding string. The letter I corresponds to a base quality score of 40 indicating a very high quality base with error probability of 0.0001.

--label_suffix string

When using --fastx_revcomp or --fastq_mergepairs, add the suffix string to sequence headers.

--maxsize positive integer

When using --fastq_filter or --fastx_filter, discard sequences with an abundance higher than the specified value.

--minsize positive integer

When using --fastq_filter or --fastx_filter, discard sequences with an abundance lower than the specified value.

--output filename

When using --fastq_eestats or --fastq_eestats2, write tabulated results to filename. See --fastq_eestats’s and --fastq_eestats2’s documentation for a complete description of the table.

--relabel_keep

When using --relabel, keep the old identifier in the header after a space.

--relabel string

Please see the description of the same option under Chimera detection for details.

--relabel_md5

Please see the description of the same option under Chimera detection for details.

--relabel_sha1

Please see the description of the same option under Chimera detection for details.

--reverse filename

When using --fastq_filter, --fastx_filter, --fastq_mergepairs or --fastq_join, specify the FASTQ file containing containing the reverse reads.

--sff_convert filename

Convert the given SFF file to FASTQ. The FASTQ output file is specified with the --fastqout option. The sequence may be clipped as specified in the SFF file if the option --sff_clip is specified, otherwise no clipping occurs. Bases that would have been clipped are converted to lower case, while the rest is in upper case. The output quality encoding may be specified with the --fastq_asciiout option (default 33). The minimum and maximum output quality scores may be limited using the --fastq_qminout and --fastq_qmaxout options.

--sff_clip

Specifies that the sequences converted by the --sff_convert command should be clipped in both ends as indicated in the SFF file. By default no clipping is performed.

	--xsize		Strip abundance information from the headers when writing the output file.
	--xee		Strip information about expected errors (ee) from the output file headers. This information is added by the --fastq_eeout and --eeout options.

Masking options:

An input sequence can be composed of lower- or uppercase letters. When soft masking is specified, lower case letters are treated as symbols that should be masked. Otherwise the case of the input sequences is ignored.

Masking is performed by the commands for chimera detection (uchime_denovo, uchime_ref), clustering (cluster_fast, cluster_smallmem, cluster_size), masking (maskfasta, fastx_mask), pairwise alignment (allpairs_global) and searching (search_exact, usearch_global).

Masking is usually specified with the --qmask option, while the --dbmask option is used for the database sequences specified with the --db option with the --usearch_global, --search_exact and --uchime_ref commands.

The argument to the --qmask and --dbmask option may be none, soft or dust. If the argument is none, the no masking is performed. If the argument is soft the lower case symbols are masked. Finally, if the argument is dust, the sequence is masked using the DUST algorithm by Tatusov and Lipman to mask low-complexity regions.

If the --hardmask option is specified, all masked regions are converted to N’s, otherwise masked regions are indicated by lower case letters.

If any sequence is masked, the masked version of the sequence (with lower case letters or N’s) is used in all output files. Otherwise the sequence is unmodified. The exception is the sequences in the output file specified with the --uchimealns option, where the input sequences are converted to upper case first and lower case letters indicate disagreement between the aligned sequences.

The --qmask option (or --dbmask for database sequences) may be combined with the --hardmask option. The results of using the none, dust or soft argument to --qmask or --dbmask are presented below, assuming each input sequence contains both lower and uppercase symbols.

Results if the --hardmask option is off (default):

	none:		no masking, all symbols used, no change
	dust:		masked symbols lowercased, rest uppercased
	soft:		lowercase symbols masked, no case changes

Results if the --hardmask option is on:

	none:		no masking, all symbols used, no change
	dust:		masked symbols changed to Ns, rest unchanged
	soft:		lowercase symbols masked and changed to Ns

When a sequence region is masked, words in the region are not included in the indices used in the heuristic search algorithm. In all other aspects, the region is treated as other regions.

Regions in sequences that are hardmasked (with N’s) have a zero alignment score and do not contribute to an alignment.

--fastaout filename

Write the masked sequences to filename, in fasta format. Applies only to the --fastx_mask command.

--fastqout filename

Write the masked sequences to filename, in fastq format. Applies only to the --fastx_mask command.

--fastx_mask filename

Mask regions in sequences contained in the specified fasta or fastq file. The default is to mask using DUST (use --qmask to modify that behavior). The output files are specified with the --fastaout and --fastqout options. The minimum and maximum percentage of unmasked residues may be specified with the --min_unmasked_pct and --max_unmasked_pct options, respectively.

--hardmask

Symbols in masked regions are replaced by N’s. The default is to replace the masked regions by lower case letters.

--maskfasta filename

Mask regions in sequences contained in the fasta file filename. The default is to mask using dust (use --qmask to modify that behavior). The output file is specified with the --output option. This command is depreciated, please use --fastx_mask instead.

--max_unmasked_pct real

Discard sequences with more than the specified maximum percentage of unmasked residues. Works only with --fastx_mask.

--min_unmasked_pct real

Discard sequences with less than the specified minimum percentage of unmasked residues. Works only with --fastx_mask.

--output filename

Write the masked sequences to filename, in fasta format. Applies only to the --mask_fasta command.

--qmask none|dust|soft

If the argument is dust, mask regions in sequences using the DUST algorithm that detects simple repeats and low-complexity regions. This is the default. If the argument is soft, mask the lower case letters in the input sequence. If the argument is none, do not mask.

Restriction site cutting options:

The input sequences in the file specified with the --cut command are cut into fragments at all restriction sites matching the pattern given with the --cut_pattern option. The fragments on the forward strand are written to the file specified with the --fastaout file and the fragments on the reverse strand are written to the file specified with the --fastaout_rev option. Input sequences that do not match are written to the file specified with the option --fastaout_discarded, and their reverse complement are also written to the file specfied with the --fastaout_discarded_rev option. The relabel options (--relabel, --relabel_keep, --relabel_md5, and --relabel_sha1) may be used to relabel the output sequences).

--cut filename

Specify the input file with sequences in FASTA format.

--cut_pattern string

Specify the restriction site cutting pattern and positions. The pattern is a string of lower- or uppercase letters specifying the nucleotides that must match, and may include ambiguous nucleotide symbols. The special characters "^" (circumflex) and "_" (underscore) are used to indicate the cutting position on the forward and reverse strand, respectively. For example, the pattern "G^AATT_C" is the pattern for the EcoRI restriction site. For such palindromic patterns (identical to its reverse complement) the command will output all possible fragments on both strands. For non-palindromic sites, it may be necessary to run the command also on the reverse complemented input sequences. Exactly one cutting site on each strand must be indicated.

--fastaout filename

Specify the output file for the resulting fragments on the forward strand.

--fastaout_rev filename

Specify the output file for the resulting fragments on the reverse strand.

--fastaout_discarded filename

Specify the output file for the non-matching sequences.

--fastaout_discarded_rev filename

Specify the output file for the non-matching seqeunces, reverse complemented.

Pairwise alignment options:

The results of the n * (n - 1) / 2 pairwise alignments are written to the result files specified with --alnout, --blast6out, --fastapairs --matched, --notmatched, --samout, --uc or --userout (see Searching section below). Specify either the --acceptall option to output all pairwise alignments, or specify an identity level with --id to discard weak alignments. Most other accept/reject options (see Searching options below) may also be used. Sequences are aligned on their plus strand only. Masking is performed as usual and specified with --qmask and --hardmask.

--acceptall

Write the results of all alignments to output files. This option overrides all other accept/reject options (including --id).

--allpairs_global filename

Perform optimal global pairwise alignments of all vs. all fasta sequences contained in filename. This command is multi-threaded.

--id real

Reject the sequence match if the pairwise identity is lower than real (value ranging from 0.0 to 1.0 included).

--threads positive integer

--uc filename

Output pairwise alignment results in filename using a tab-separated uclust-like format with 10 columns. Each sequence is compared to all other sequences, and all hits (--acceptall) or only some hits (--id float) are reported, with one pairwise comparison per line:

	1.		Record type, always set to ’H’.
	2.		Ordinal number of the target sequence (based on input order, starting from zero).
	3.		Sequence length.
	4.		Percentage of similarity with the target sequence.
	5.		Match orientation, always set to ’+’.
	6.		Not used, always set to zero.
	7.		Not used, always set to zero.
	8.		Compact representation of the pairwise alignment using the CIGAR format (Compact Idiosyncratic Gapped Alignment Report): M (match/mismatch), D (deletion) and I (insertion). The equal sign ’=’ indicates that the query is identical to the centroid sequence.
	9.		Label of the query sequence.
	10.		Label of the target sequence.

Searching options:

--alnout filename

Write pairwise global alignments to filename using a human-readable format. Use --rowlen to modify alignment length. Output order may vary when using multiple threads.

--biomout filename

Write search results to an OTU table in the biom version 1.0 file format. The query file contains the samples, while the database file contains the OTUs. Sample and OTU identifiers are extracted from the header of these sequences. See the --biomout option in the Clustering section for further details.

--blast6out filename

Write search results to filename using a blast-like tab-separated format of twelve fields (listed below), with one line per query-target matching (or lack of matching if --output_no_hits is used). Warning, vsearch uses global pairwise alignments, not blast’s seed-and-extend algorithm. Therefore, some common blast output values (alignment start and end, evalue, bit score) are reported differently. Output order may vary when using multiple threads. A similar output can be obtain with --userout filename and --userfields query+target+id+alnlen+mism+opens+qlo+qhi+tlo+thi+evalue+bits. A complete list and description is available in the section ’Userfields’ of this manual.

	1.	query: query label.
	2.	target: target (database sequence) label. The field is set to ’*’ if there is no alignment.
	3.	id: percentage of identity (real value ranging from 0.0 to 100.0). The percentage identity is defined as 100 * (matching columns) / (alignment length - terminal gaps). See fields id0 to id4 for other definitions.
	4.	alnlen: length of the query-target alignment (number of columns). The field is set to 0 if there is no alignment.
	5.	mism: number of mismatches in the alignment (zero or positive integer value).
	6.	opens: number of columns containing a gap opening (zero or positive integer value).
	7.	qlo: first nucleotide of the query aligned with the target. Always equal to 1 if there is an alignment, 0 otherwise (see qilo to ignore initial gaps).
	8.	qhi: last nucleotide of the query aligned with the target. Always equal to the length of the pairwise alignment, 0 otherwise (see qihi to ignore terminal gaps).
	9.	tlo: first nucleotide of the target aligned with the query. Always equal to 1 if there is an alignment, 0 otherwise (see tilo to ignore initial gaps).
	10.	thi: last nucleotide of the target aligned with the query. Always equal to the length of the pairwise alignment, 0 otherwise (see tihi to ignore terminal gaps).
	11.	evalue: expectancy-value (not computed for nucleotide alignments). Always set to -1.
	12.	bits: bit score (not computed for nucleotide alignments). Always set to 0.

--db filename

Compare query sequences (specified with --usearch_global) to the fasta-formatted target sequences contained in filename, using global pairwise alignment. Alternatively, the name of a preformatted UDB database created using the makeudb_usearch command (see below) may be specified.

--dbmask none|dust|soft

Mask regions in the target database sequences using the dust method or the soft method, or do not mask (none). Warning, when using soft masking search commands become case sensitive. The default is to mask using dust.

--dbmatched filename

Write database target sequences matching at least one query sequence to filename, in fasta format. If the option --sizeout is used, the number of queries that matched each target sequence is indicated using the pattern ";size=integer;".

--dbnotmatched filename

Write database target sequences not matching query sequences to filename, in fasta format.

--fastapairs filename

Write pairwise alignments of query and target sequences to filename, in fasta format.

--fulldp

Dummy option for compatibility with usearch. To maximize search sensitivity, vsearch uses a 8-way 16-bit SIMD vectorized full dynamic programming algorithm (Needleman-Wunsch), whether or not --fulldp is specified.

--gapext string

Set penalties for a gap extension. See --gapopen for a complete description of the penalty declaration system. The default is to initialize the six gap extending penalties using a penalty of 2 for extending internal gaps and a penalty of 1 for extending terminal gaps, in both query and target sequences (i.e. 2I/1E).

--gapopen string

Set penalties for a gap opening. A gap opening can occur in six different contexts: in the query (Q) or in the target (T) sequence, at the left (L) or right (R) extremity of the sequence, or inside the sequence (I). Sequence symbols (Q and T) can be combined with location symbols (L, I, and R), and numerical values to declare penalties for all possible contexts: aQL/bQI/cQR/dTL/eTI/fTR, where abcdef are zero or positive integers, and ’/’ is used as a separator.
To simplify declarations, the location symbols (L, I, and R) can be combined, the symbol (E) can be used to treat both extremities (L and R) equally, and the symbols Q and T can be omitted to treat query and target sequences equally. For instance, the default is to declare a penalty of 20 for opening internal gaps and a penalty of 2 for opening terminal gaps (left or right), in both query and target sequences (i.e. 20I/2E). If only a numerical value is given, without any sequence or location symbol, then the penalty applies to all gap openings. To forbid gap-opening, an infinite penalty value can be declared with the symbol ’*’. To use vsearch as a semi-global aligner, a null-penalty can be applied to the left (L) or right (R) gaps.
vsearch always initializes the six gap opening penalties using the default parameters (20I/2E). The user is then free to declare only the values he/she wants to modify. The string is scanned from left to right, accepted symbols are (0123456789/LIREQT*), and later values override previous values.
Please note that vsearch, in contrast to usearch, only allows integer gap penalties. Because the lowest gap penalties are 0.5 by default in usearch, all default scores and gap penalties in vsearch have been doubled to maintain equivalent penalties and to produce identical alignments.

--hardmask

Mask sequence regions by replacing them with Ns instead of setting them to lower case as is the default. For more information, please see the Masking section.

--id real

Reject the sequence match if the pairwise identity is lower than real (value ranging from 0.0 to 1.0 included). The search process sorts target sequences by decreasing number of k-mers they have in common with the query sequence, using that information as a proxy for sequence similarity. That efficient pre-filtering also prevents pairwise alignments with weakly matching targets, as there needs to be at least 6 shared k-mers to start the pairwise alignment, and at least one out of every 16 k-mers from the query needs to match the target. Consequently, using values lower than --id 0.5 is not likely to capture more weakly matching targets. The pairwise identity is by default defined as the number of (matching columns) / (alignment length - terminal gaps). That definition can be modified by --iddef.

--iddef 0|1|2|3|4

Change the pairwise identity definition used in --id. Values accepted are:

	0.	CD-HIT definition: (matching columns) / (shortest sequence length).
	1.	edit distance: (matching columns) / (alignment length).
	2.	edit distance excluding terminal gaps (default definition for --id).
	3.	Marine Biological Lab definition counting each gap opening (internal or terminal) as a single mismatch, whether or not the gap was extended: 1.0 - [(mismatches + gap openings)/(longest sequence length)]
	4.	BLAST definition, equivalent to --iddef 1 for global pairwise alignments.

The option --userfields accepts the fields id0 to id4, in addition to the field id, to report the pairwise identity values corresponding to the different definitions.

--idprefix positive integer

Reject the sequence match if the first integer nucleotides of the target do not match the query.

--idsuffix positive integer

Reject the sequence match if the last integer nucleotides of the target do not match the query.

--leftjust

Reject the sequence match if the pairwise alignment begins with gaps.

--match integer

Score assigned to a match (i.e. identical nucleotides) in the pairwise alignment. The default value is 2.

--matched filename

Write query sequences matching database target sequences to filename, in fasta format.

--maxaccepts positive integer

Maximum number of hits to accept before stopping the search. The default value is 1. This option works in pair with --maxrejects. The search process sorts target sequences by decreasing number of k-mers they have in common with the query sequence, using that information as a proxy for sequence similarity. After pairwise alignments, if the first target sequence passes the acceptation criteria, it is accepted as best hit and the search process stops for that query. If --maxaccepts is set to a higher value, more hits are accepted. If --maxaccepts and --maxrejects are both set to 0, the complete database is searched.

--maxdiffs positive integer

Reject the sequence match if the alignment contains at least integer substitutions, insertions or deletions.

--maxgaps positive integer

Reject the sequence match if the alignment contains at least integer insertions or deletions.

--maxhits positive integer

Maximum number of hits to show once the search is terminated (hits are sorted by decreasing identity). Unlimited by default. That option applies to --alnout, --blast6out, --fastapairs, --samout, --uc, or --userout output files.

--maxid real

Reject the sequence match if the percentage of identity between the two sequences is greater than real.

--maxqsize positive integer

Reject query sequences with an abundance greater than integer.

--maxqt real

Reject if the query/target sequence length ratio is greater than real.

--maxrejects positive integer

Maximum number of non-matching target sequences to consider before stopping the search. The default value is 32. This option works in pair with --maxaccepts. The search process sorts target sequences by decreasing number of k-mers they have in common with the query sequence, using that information as a proxy for sequence similarity. After pairwise alignments, if none of the first 32 examined target sequences pass the acceptation criteria, the search process stops for that query (no hit). If --maxrejects is set to a higher value, more target sequences are considered. If --maxaccepts and --maxrejects are both set to 0, the complete database is searched.

--maxsizeratio real

Reject if the query/target abundance ratio is greater than real.

--maxsl real

Reject if the shorter/longer sequence length ratio is greater than real.

--maxsubs positive integer

Reject the sequence match if the pairwise alignment contains more than integer substitutions.

--mid real

Reject the sequence match if the percentage of identity is lower than real (ignoring all gaps, internal and terminal).

--mincols positive integer

Reject the sequence match if the alignment length is shorter than integer.

--minqt real

Reject if the query/target sequence length ratio is lower than real.

--minsizeratio real

Reject if the query/target abundance ratio is lower than real.

--minsl real

Reject if the shorter/longer sequence length ratio is lower than real.

--mintsize positive integer

Reject target sequences with an abundance lower than integer.

--minwordmatches non-negative integer

Minimum number of word matches required for a sequence to be considered further. Default value is 12 for the default word length 8. For word lengths 3-15, the default minimum word matches are 18, 17, 16, 15, 14, 12, 11, 10, 9, 8, 7, 5 and 3, respectively. If the query sequence has fewer unique words than the number specified, all words in the query must match. If the argument is 0, no word matches are required.

--mismatch integer

Score assigned to a mismatch (i.e. different nucleotides) in the pairwise alignment. The default value is -4.

--mothur_shared_out filename

Write search results to an OTU table in the mothur ’shared’ tab-separated plain text file format. The query file contains the samples, while the database file contains the OTUs. Sample and OTU identifiers are extracted from the header of these sequences. See the --otutabout option in the Clustering section for further details.

--notmatched filename

Write query sequences not matching database target sequences to filename, in fasta format.

--otutabout filename

Write search results to an OTU table in the classic tab-separated plain text format. The query file contains the samples, while the database file contains the OTUs. Sample and OTU identifiers are extracted from the header of these sequences. See the --mothur_shared_out option in the Clustering section for further details.

--output_no_hits

Write both matching and non-matching queries to --alnout, --blast6out, --samout or --userout output files. Non-matching queries are labelled ’No hits’ in --alnout files.

--pattern string

This option is ignored. It is provided for compatibility with usearch.

--qmask none|dust|soft

Mask regions in the query sequences using the dust or the soft algorithms, or do not mask (none). Warning, when using soft masking search commands become case sensitive. The default is to mask using dust.

--query_cov real

Reject if the fraction of the query aligned to the target sequence is lower than real. The query coverage is computed as (matches + mismatches) / query sequence length. Internal or terminal gaps are not taken into account.

--rightjust

Reject the sequence match if the pairwise alignment ends with gaps.

--rowlen positive integer

Width of alignment lines in --alnout output. The default value is 64. Set to 0 to eliminate wrapping.

--samheader

Include header lines to the SAM file when --samout is specified. The header includes lines starting with @HD, @SQ and @PG, but no @RG lines (see (link) <https://github.com/samtools/hts-specs>). By default no header line is written.

--samout filename

Write alignment results to filename using the SAM format (a tab-separated text file). When using the --samheader option, the SAM file starts with header lines. Each non-header line is a SAM record, which represents either a query-target alignment or the absence of match for a query (output order may vary when using multiple threads). Each record contains 11 mandatory fields and optional fields (see (link) <https://github.com/samtools/hts-specs> for a complete description of the format):

	1.	query sequence label.
	2.	combination of bitwise flags. Possible values are: 0 (top hit), 4 (no hit), 16 (reverse-complemented hit), 256 (secondary hit, i.e. all hits except the top hit).
	3.	target sequence label.
	4.	first position of a target aligned with the query (always 1 for global pairwise alignments, 0 if there is no match).
	5.	mapping quality (ignored, always set to ’*’).
	6.	CIGAR string (set to ’*’ if there is no match).
	7.	name of the target sequence matching with the next read of the query (for mate reads only, ignored and always set to ’*’).
	8.	position of the primary alignment of the next read of the query (for mate reads only, ignored and always set to 0).
	9.	target sequence length (for multi-segment targets, ignored and always set to 0).
	10.	query sequence (complete, not only the segment aligned to the target as usearch does).
	11.	quality string (ignored, always set to ’*’).

Optional fields for query-target matches (number and order of fields may vary):

12.

AS:i:? alignment score (i.e.

percentage of identity).

	13.	XN:i:? next best alignment score (always set to 0).
	14.	XM:i:? number of mismatches.
	15.	XO:i:? number of gap openings (excluding terminal gaps).

	16.		XG:i:? number of gap extensions (excluding terminal gaps).
	17.		NM:i:? edit distance to the target (sum of XM and XG).

	18.	MD:Z:? string for mismatching positions.
	19.	YT:Z:UU string representing the alignment type.

--search_exact filename

Search for exact full-length matches to the query sequences contained in filename in the database of target sequences (--db). Only 100% exact matches are reported and this command is much faster than --usearch_global. The --id, --maxaccepts and --maxrejects options are ignored, but the rest of the searching options may be specified.

	--self		Reject the sequence match if the query and target labels are identical.
	--selfid		Reject the sequence match if the query and target sequences are strictly identical.

--sizeout

Add abundance annotations to the output of the option --dbmatched (using the pattern ’;size=integer;’), to report the number of queries that matched each target.

--strand plus|both

When searching for similar sequences, check the plus strand only (default) or check both strands.

--target_cov real

Reject the sequence match if the fraction of the target sequence aligned to the query sequence is lower than real. The target coverage is computed as (matches + mismatches) / target sequence length. Internal or terminal gaps are not taken into account.

--top_hits_only

Only the top hits between the query and database sequence sets are written to the output specified with the options --alnout, --samout, --userout, --blast6out, --uc, --fastapairs, --matched or --notmatched (but not --dbmatched and --dbnotmatched). For each query, the top hit is the one presenting the highest percentage of identity (see the --iddef option to change the way identity is measured). For a given query, if several top hits present exactly the same percentage of identity, the number of hits reported is controlled by the --maxaccepts value (1 by default).

--uc filename

Output searching results in filename using a tab-separated uclust-like format with 10 columns. When using the --search_exact command, the table layout is the same than with the --allpairs_global. When using the --usearch_global command, the table present two different type of entries: hit (H) or no hit (N). Each query sequence is compared to all other sequences, and the best hit (--maxaccept 1) or several hits (--maxaccept > 1) are reported (H). Output order may vary when using multiple threads. Column content varies with the type of entry (H or N):

	1.		Record type: H, or N (’hit’ or ’no hit’).
	2.		Ordinal number of the target sequence (based on input order, starting from zero). Set to ’*’ for N.
	3.		Sequence length. Set to ’*’ for N.
	4.		Percentage of similarity with the target sequence. Set to ’*’ for N.
	5.		Match orientation + or -. . Set to ’.’ for N.
	6.		Not used, always set to zero for H, or ’*’ for N.
	7.		Not used, always set to zero for H, or ’*’ for N.
	8.		Compact representation of the pairwise alignment using the CIGAR format (Compact Idiosyncratic Gapped Alignment Report): M (match/mismatch), D (deletion) and I (insertion). The equal sign ’=’ indicates that the query is identical to the centroid sequence. Set to ’*’ for N.
	9.		Label of the query sequence.
	10.		Label of the target centroid sequence. Set to ’*’ for N.

--uc_allhits

When using the --uc option, show all hits, not just the top hit for each query.

--usearch_global filename

Compare target sequences (--db) to the fasta-formatted query sequences contained in filename, using global pairwise alignment.

--userfields string

When using --userout, select and order the fields written to the output file. Fields are separated by ’+’ (e.g. query+target+id). See the ’Userfields’ section for a complete list of fields.

--userout filename

Write user-defined tab-separated output to filename. Select the fields with the option --userfields. Output order may vary when using multiple threads. If --userfields is empty or not present, filename is empty.

--weak_id real

Show hits with percentage of identity of at least real, without terminating the search. A normal search stops as soon as enough hits are found (as defined by --maxaccepts, --maxrejects, and --id). As --weak_id reports weak hits that are not deduced from --maxaccepts, high --id values can be used, hence preserving both speed and sensitivity. Logically, real must be smaller than the value indicated by --id.

--wordlength positive integer

Length of words (i.e. k-mers) for database indexing. The range of possible values goes from 3 to 15, but values near 8 or 9 are generally recommended. Longer words may reduce the sensitivity/recall for weak similarities, but can increase precision. On the other hand, shorter words may increase sensitivity or recall, but may reduce precision. Computation time generally increases with shorter words and decreases with longer words, but it increases again for very long words. Memory requirements for a part of the index increase with a factor of 4 each time word length increases by one nucleotide, and this generally becomes significant for long words (12 or more). The default value is 8.

Shuffling options:

Fasta entries in the input file are outputted in a pseudo-random order.

--output filename

Write the shuffled sequences to filename, in fasta format.

--randseed positive integer

When shuffling sequence order, use integer as seed. A given seed always produces the same output order (useful for replicability). Set to 0 to use a pseudo-random seed (default behavior).

--relabel string

Relabel sequences using the prefix string and a ticker (1, 2, 3, etc.) to construct the new headers. Use --sizeout to conserve the abundance annotations.

--relabel_keep

When relabelling, keep the old identifier in the header after a space.

--relabel_md5

Relabel sequences using the MD5 message digest algorithm applied to each sequence. Former sequence headers are discarded. The sequence is converted to upper case and U is replaced by T before the digest is computed. The MD5 digest is a cryptographic hash function designed to minimize the probability that two different inputs gives the same output, even for very similar, but non-identical inputs. Still, there is always a very small, but non-zero probability that two different inputs give the same result. The MD5 digest generates a 128-bit (16-byte) digest that is represented by 16 hexadecimal numbers (using 32 symbols among 0123456789abcdef). Use --sizeout to conserve the abundance annotations.

--relabel_sha1

Relabel sequences using the SHA1 message digest algorithm applied to each sequence. It is similar to the --relabel_md5 option but uses the SHA1 algorithm instead of the MD5 algorithm. The SHA1 digest generates a 160-bit (20-byte) result that is represented by 20 hexadecimal numbers (40 symbols). The probability of a collision (two non-identical sequences having the same digest) is smaller for the SHA1 algorithm than it is for the MD5 algorithm. Use --sizeout to conserve the abundance annotations.

--sizeout

When using --relabel, --relabel_md5 or --relabel_sha1, preserve and report abundance annotations to the output fasta file (using the pattern ’;size=integer;’).

--shuffle filename

Pseudo-randomly shuffle the order of sequences contained in filename.

--topn positive integer

Output only the first integer sequences after pseudo-random reordering.

--xsize

Strip abundance information from the headers when writing the output file.

Sorting options:

Fasta entries are sorted by decreasing abundance (--sortbysize) or sequence length (--sortbylength). To obtain a stable sorting order, ties are sorted by decreasing abundance and label increasing alpha-numerical order (--sortbylength), or just by label increasing alpha-numerical order (--sortbysize). Label sorting assumes that all sequences have unique labels. The same applies to the automatic sorting performed during chimera checking (--uchime_denovo), dereplication (--derep_fulllength), and clustering (--cluster_fast and --cluster_size).

--maxsize positive integer

When using --sortbysize, discard sequences with an abundance value greater than integer.

--minsize positive integer

When using --sortbysize, discard sequences with an abundance value smaller than integer.

--output filename

Write the sorted sequences to filename, in fasta format.

--relabel string

Please see the description of the same option under Chimera detection for details.

--relabel_keep

When relabelling, keep the old identifier in the header after a space.

--relabel_md5

Please see the description of the same option under Chimera detection for details.

--relabel_sha1

Please see the description of the same option under Chimera detection for details.

--sizeout

When using --relabel, report abundance annotations to the output fasta file (using the pattern ’;size=integer;’).

--sortbylength filename

Sort by decreasing length the sequences contained in filename. See the general options --minseqlength and --maxseqlength to eliminate short and long sequences.

--sortbysize filename

Sort by decreasing abundance the sequences contained in filename (missing abundance values are assumed to be ’;size=1’). See the options --minsize and --maxsize to eliminate rare and dominant sequences.

--topn positive integer

Output only the top integer sequences (i.e. the longest or the most abundant).

--xsize

Strip abundance information from the headers when writing the output file.

Subsampling options:

Subsampling randomly extracts a certain number or a certain percentage of the sequences in the input file. If the --sizein option is in effect, the abundances of the input sequences is taken into account and the sampling is performed as if the input sequences were rereplicated, subsampled and dereplicated before being written to the output file. The extraction is performed as a random sampling with a uniform distribution among the input sequences and is performed without replacement. The input file is specified with the --fastx_subsample option, the output files are specified with the --fastaout and --fastqout options and the amount of sequences to be sampled is specified with the --sample_pct or --sample_size options. The sequences not sampled may be written to files specified with the options --fasta_discarded and --fastq_discarded. The --fastq_ascii, --fastq_qmin and --fastq_qmax options are also available.

--fastaout filename

Write the sampled sequences to filename, in fasta format.

--fastaout_discarded filename

Write the sequences not sampled to filename, in fasta format.

--fastq_ascii positive integer

--fastq_qmax positive integer

Specify the maximum quality score accepted when reading FASTQ files. The default is 41, which is usual for recent Sanger/Illumina 1.8+ files.

--fastq_qmin positive integer

Specify the minimum quality score accepted for FASTQ files. The default is 0, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may use scores between -5 and 2.

--fastqout filename

Write the sampled sequences to filename, in fastq format. Requires input in fastq format.

--fastqout_discarded filename

Write the sequences not sampled to filename, in fastq format. Requires input in fastq format.

--fastx_subsample filename

Perform subsampling from the sequences in the specified input file that is in FASTA or FASTQ format.

--randseed positive integer

Use integer as a seed for the pseudo-random generator. A given seed always produces the same output, which is useful for replicability. Set to 0 to use a pseudo-random seed (default behavior).

--relabel string

Relabel sequences using the prefix string and a ticker (1, 2, 3, etc.) to construct the new headers. Use --sizeout to conserve the abundance annotations.

--relabel_keep

When relabelling, keep the old identifier in the header after a space.

--relabel_md5

Relabel sequences using the MD5 message digest algorithm applied to each sequence. Former sequence headers are discarded. The sequence is converted to upper case and U is replaced by T before the digest is computed. The MD5 digest is a cryptographic hash function designed to minimize the probability that two different inputs give the same output, even for very similar, but non-identical inputs. Still, there is always a very small, but non-zero probability that two different inputs give the same result. The MD5 digest generates a 128-bit (16-byte) digest that is represented by 16 hexadecimal numbers (using 32 symbols among 0123456789abcdef). Use --sizeout to conserve the abundance annotations.

--relabel_sha1

Relabel sequences using the SHA1 message digest algorithm applied to each sequence. It is similar to the --relabel_md5 option but uses the SHA1 algorithm instead of the MD5 algorithm. The SHA1 digest generates a 160-bit (20-byte) result that is represented by 20 hexadecimal numbers (40 symbols). The probability of a collision (two non-identical sequences having the same digest) is smaller for the SHA1 algorithm than it is for the MD5 algorithm. Use --sizeout to conserve the abundance annotations.

--sample_pct real

Subsample the given percentage of the input sequences. Accepted values range from 0.0 to 100.0.

--sample_size positive integer

Extract the given number of sequences.

--sizein

Take the abundance information of the input file into account, otherwise the abundance of each sequence is considered to be 1.

--sizeout

Write abundance information to the output file.

--xsize

Strip abundance information from the headers when writing the output file.

Taxonomic classification options:

The vsearch command --sintax will classify the input sequences according to the Sintax algorithm as described by Robert Edgar (2016) in SINTAX: a simple non-Bayesian taxonomy classifier for 16S and ITS sequences, BioRxiv, 074161. Preprint. doi: 10.1101/074161 (link)

The name of the fasta file containing the input sequences to be classified is given as an argument to the --sintax command. The reference sequence database is specified with the --db option. The results are written in a tab delimited text file whose name is specified with the --tabbedout option. The --sintax_cutoff option may be used to set a minimum level of bootstrap support for the taxonomic ranks to be reported.

Multithreading is supported. Databases in UDB files are supported. The strand option may be specified.

The reference database must contain taxonomic information in the header of each sequence in the form of a string starting with ";tax=" and followed by a comma-separated list of up to eight taxonomic identifiers. Each taxonomic identifier must start with an indication of the rank by one of the letters d (for domain) k (kingdom), p (phylum), c (class), o (order), f (family), g (genus), or s (species). The letter is followed by a colon (:) and the name of that rank. Commas and semicolons are not allowed in the name of the rank.

Example: ">X80725_S000004313;tax=d:Bacteria,p:Proteobacteria, c:Gammaproteobacteria,o:Enterobacteriales,f:Enterobacteriaceae, g:Escherichia/Shigella,s:Escherichia_coli".

--db filename

Read the reference sequences from filename, in FASTA, FASTQ or UDB format. These sequences needs to be annotated with taxonomy.

--sintax_cutoff real

Specify a minimum level of bootstrap support for the taxonomic ranks that will be included in column 4 of the output file. For instance 0.9, corresponding to 90%.

--sintax filename

Read the input sequences from filename, in FASTA or FASTQ format.

--tabbedout filename

Write the results to filename, in a tab-separated text format. Column 1 contains the query label. Column 2 contains the predicted taxonomy in the same format as for the reference data, with bootstrap support indicated in parentheses after each rank. Column 3 contains the strand. If the --sintax_cutoff option is used, the predicted taxonomy will be repeated in column 4 while omitting the bootstrap values and including only the ranks with support at or above the threshold.

UDB options:

Databases to be used with the --usearch_global command may be prepared from FASTA files and stored to a binary UDB formatted file in order to speed up searching. This may be worthwhile when searching a large database repeatedly. The sequences are indexed and stored in a way that can be quickly loaded into memory. The commands and options below can be used to create and inspect UDB files. An UDB file may be specified with the --db option instead of a FASTA formatted file with the --usearch_global command.

--dbmask none|dust|soft

Specify the sequence masking method used with the --makeudb_usearch command, either none, dust or soft. No masking is performed when none is specified. When dust is specified, the DUST algorithm will be used for masking low complexity regions (short repeats and skewed composition). Lower case letters in the input file will be masked when soft is specified (soft masking).

--hardmask

Mask sequences by replacing letters with N for the --makeudb_usearch command. The default is to use lower case letters (soft masking).

--makeudb_usearch filename

Create an UDB database file from the FASTA-formatted sequences in the file with the given filename. The UDB database is written to the file specified with the --output option.

--output filename

Specify the filename of a FASTA or UDB output file for the --makeudb_usearch or the --udb2fasta command, respectively.

--udb2fasta filename

Read the UDB database in the file with the given filename and output the sequences in FASTA format in the file specified by the --output option.

--udbinfo filename

Show information about the UDB database in the file with the given filename.

--udbstats filename

Report statistics about the indexed words in the UDB database in the file with the given filename.

--wordlength positive integer

Specify the length of the words to be used when creating the UDB database index using the --makeudb_usearch command. Valid numbers range from 3 to 15. The default is 8.

Userfields (fields accepted by the --userfields option):

	aln	Print a string of M (match), D (delete, i.e. a gap in the query) and I (insert, i.e. a gap in the target) representing the pairwise alignment. Empty field if there is no alignment.
	alnlen	Print the length of the query-target alignment (number of columns). The field is set to 0 if there is no alignment.
	bits	Bit score (not computed for nucleotide alignments). Always set to 0.
	caln	Compact representation of the pairwise alignment using the CIGAR format (Compact Idiosyncratic Gapped Alignment Report): M (match/mismatch), D (deletion) and I (insertion). Empty field if there is no alignment.
	evalue	E-value (not computed for nucleotide alignments). Always set to -1.
	exts	Number of columns containing a gap extension (zero or positive integer value).
	gaps	Number of columns containing a gap (zero or positive integer value).
	id	Percentage of identity (real value ranging from 0.0 to 100.0). The percentage identity is defined as 100 * (matching columns) / (alignment length - terminal gaps).
	id0	CD-HIT definition of the percentage of identity (real value ranging from 0.0 to 100.0) using the length of the shortest sequence in the pairwise alignment as denominator: 100 * (matching columns) / (shortest sequence length).
	id1	The percentage of identity (real value ranging from 0.0 to 100.0) is defined as the edit distance: 100 * (matching columns) / (alignment length).
	id2	The percentage of identity (real value ranging from 0.0 to 100.0) is defined as the edit distance, excluding terminal gaps. The field id2 is an alias for the field id.
	id3	Marine Biological Lab definition of the percentage of identity (real value ranging from 0.0 to 100.0), counting each gap opening (internal or terminal) as a single mismatch, whether or not the gap was extended, and using the length of the longest sequence in the pairwise alignment as denominator: 100 * (1.0 - [(mismatches + gaps) / (longest sequence length)]).
	id4	BLAST definition of the percentage of identity (real value ranging from 0.0 to 100.0), equivalent to --iddef 1 in a context of global pairwise alignment. The field id4 is always equal to the field id1.
	ids	Number of matches in the alignment (zero or positive integer value).
	mism	Number of mismatches in the alignment (zero or positive integer value).
	opens	Number of columns containing a gap opening (zero or positive integer value).
	pairs	Number of columns containing only nucleotides. That value corresponds to the length of the alignment minus the gap-containing columns (zero or positive integer value).
	pctgaps	Number of columns containing gaps expressed as a percentage of the alignment length (real value ranging from 0.0 to 100.0).
	pctpv	Percentage of positive columns. When working with nucleotide sequences, this is equivalent to the percentage of matches (real value ranging from 0.0 to 100.0).
	pv	Number of positive columns. When working with nucleotide sequences, this is equivalent to the number of matches (zero or positive integer value).
	qcov	Fraction of the query sequence that is aligned with the target sequence (real value ranging from 0.0 to 100.0). The query coverage is computed as 100.0 * (matches + mismatches) / query sequence length. Internal or terminal gaps are not taken into account. The field is set to 0.0 if there is no alignment.
	qframe	Query frame (-3 to +3). That field only concerns coding sequences and is not computed by vsearch. Always set to +0.
	qhi	Last nucleotide of the query aligned with the target. Always equal to the length of the pairwise alignment, 0 otherwise (see qihi to ignore terminal gaps).
	qihi	Last nucleotide of the query aligned with the target (ignoring terminal gaps). Nucleotide numbering starts from 1. The field is set to 0 if there is no alignment.
	qilo	First nucleotide of the query aligned with the target (ignoring initial gaps). Nucleotide numbering starts from 1. The field is set to 0 if there is no alignment.
	ql	Query sequence length (positive integer value). The field is set to 0 if there is no alignment.
	qlo	First nucleotide of the query aligned with the target. Always equal to 1 if there is an alignment, 0 otherwise (see qilo to ignore initial gaps).
	qrow	Print the sequence of the query segment as seen in the pairwise alignment (i.e. with gap insertions if need be). Empty field if there is no alignment.
	qs	Query segment length. Always equal to query sequence length.
	qstrand	Query strand orientation (+ or - for nucleotide sequences). Empty field if there is no alignment.
	query	Query label.
	raw	Raw alignment score (negative, null or positive integer value). The score is the sum of match rewards minus mismatch penalties, gap openings and gap extensions. The field is set to 0 if there is no alignment.
	target	Target label. The field is set to ’*’ if there is no alignment.
	tcov	Fraction of the target sequence that is aligned with the query sequence (real value ranging from 0.0 to 100.0). The target coverage is computed as 100.0 * (matches + mismatches) / target sequence length. Internal or terminal gaps are not taken into account. The field is set to 0.0 if there is no alignment.
	tframe	Target frame (-3 to +3). That field only concerns coding sequences and is not computed by vsearch. Always set to +0.
	thi	Last nucleotide of the target aligned with the query. Always equal to the length of the pairwise alignment, 0 otherwise (see tihi to ignore terminal gaps).
	tihi	Last nucleotide of the target aligned with the query (ignoring terminal gaps). Nucleotide numbering starts from 1. The field is set to 0 if there is no alignment.
	tilo	First nucleotide of the target aligned with the query (ignoring initial gaps). Nucleotide numbering starts from 1. The field is set to 0 if there is no alignment.
	tl	Target sequence length (positive integer value). The field is set to 0 if there is no alignment.
	tlo	First nucleotide of the target aligned with the query. Always equal to 1 if there is an alignment, 0 otherwise (see tilo to ignore initial gaps).
	trow	Print the sequence of the target segment as seen in the pairwise alignment (i.e. with gap insertions if need be). Empty field if there is no alignment.
	ts	Target segment length. Always equal to target sequence length. The field is set to 0 if there is no alignment.
	tstrand	Target strand orientation (+ or - for nucleotide sequences). Always set to ’+’, so reverse strand matches have tstrand ’+’ and qstrand

DELIBERATE CHANGES

If you are a usearch user, our objective is to make you feel at home. That’s why vsearch was designed to behave like usearch, to some extent. Like any complex software, usearch is not free from quirks and inconsistencies. We decided not to reproduce some of them, and for complete transparency, to document here the deliberate changes we made.

During a search with usearch, when using the options --blast6out and --output_no_hits, for queries with no match the number of fields reported is 13, where it should be 12. This is corrected in vsearch.

The field raw of the --userfields option is not informative in usearch. This is corrected in vsearch.

The fields qlo, qhi, tlo, thi now have counterparts (qilo, qihi, tilo, tihi) reporting alignment coordinates ignoring terminal gaps.

In usearch, when using the option --output_no_hits, queries that receive no match are reported in --blast6out file, but not in the alignment output file. This is corrected in vsearch.

vsearch introduces a new --cluster_size command that sorts sequences by decreasing abundance before clustering.

vsearch reintroduces --iddef alternative pairwise identity definitions that were removed from usearch.

vsearch extends the --topn option to sorting commands.

vsearch extends the --sizein option to dereplication (--derep_fulllength) and clustering (--cluster_fast).

vsearch treats T and U as identical nucleotides during dereplication.

vsearch sorting is stabilized by using sequence abundances or sequences labels as secondary or tertiary keys.

vsearch by default uses the DUST algorithm for masking low-complexity regions. Masking behavior is also slightly changed to be more consistent.

NOVELTIES

vsearch introduces new commands and new options not present in usearch 7. They are described in the ’Options’ section of this manual. Here is a short list:

uchime2_denovo, uchime3_denovo, alignwidth, borderline, fasta_score (chimera checking)

	-		cluster_size, cluster_unoise, clusterout_id, clusterout_sort, profile (clustering)
	-		fasta_width, gzip_decompress, bzip2_decompress (general option)
	-		iddef (clustering, pairwise alignment, searching)
	-		maxuniquesize (dereplication)

	-		relabel_md5 and relabel_sha1 (chimera detection, dereplication, FASTQ processing, shuffling, sorting)
	-		shuffle (shuffling)
	-		fastq_eestats, fastq_eestats2, fastq_maxlen, fastq_truncee (FASTQ processing)
	-		fastaout_discarded, fastqout_discarded (subsampling)
	-		rereplicate (dereplication/rereplication)

EXAMPLES

Align all sequences in a database with each other and output all pairwise alignments:

vsearch --allpairs_global database.fas --alnout results.aln --acceptall

Check for the presence of chimeras (de novo); parents should be at least 1.5 times more abundant than chimeras. Output non-chimeric sequences in fasta format (no wrapping):

vsearch --uchime_denovo queries.fas --abskew 1.5 --nonchimeras results.fas --fasta_width 0

Cluster with a 97% similarity threshold, collect cluster centroids, and write cluster descriptions using a uclust-like format:

vsearch --cluster_fast queries.fas --id 0.97 --centroids centroids.fas --uc clusters.uc

Dereplicate the sequences contained in queries.fas, take into account the abundance information already present, write unwrapped fasta sequences to queries_unique.fas with the new abundance information, discard all sequences with an abundance of 1:

vsearch --derep_fulllength queries.fas --sizein --fasta_width 0 --sizeout --output queries_unique.fas --minuniquesize 2

Mask simple repeats and low complexity regions in the input fasta file with the DUST algorithm (masked regions are lowercased), and write the results to the output file:

vsearch --maskfasta queries.fas --qmask dust --output queries_masked.fas

Search queries in a reference database, with a 80%-similarity threshold, take terminal gaps into account when calculating pairwise similarities, output pairwise alignments:

vsearch --usearch_global queries.fas --db references.fas --id 0.8 --iddef 1 --alnout results.aln

Search a sequence dataset against itself (ignore self hits), get all matches with at least 60% similarity, and collect results in a blast-like tab-separated format. Accept an unlimited number of hits (--maxaccepts 0), and compare each query to all other sequences, including unlikely candidates (--maxrejects 0):

vsearch --usearch_global queries.fas --db queries.fas --self --id 0.6 --blast6out results.blast6 --maxaccepts 0 --maxrejects 0

Shuffle the input fasta file (change the order of sequences) in a repeatable fashion (fixed seed), and write unwrapped fasta sequences to the output file:

vsearch --shuffle queries.fas --output queries_shuffled.fas --randseed 13 --fasta_width 0

Sort by decreasing abundance the sequences contained in queries.fas (using the ’size=integer’ information), relabel the sequences while preserving the abundance information (with --sizeout), keep only sequences with an abundance equal to or greater than 2:

vsearch --sortbysize queries.fas --output queries_sorted.fas --relabel sampleA_ --sizeout --minsize 2

AUTHORS

Implementation by Torbjørn Rognes and Tomás Flouri, documentation by Frédéric Mahé.

CITATION

Rognes T, Flouri T, Nichols B, Quince C, Mahé F. (2016) VSEARCH: a versatile open source tool for metagenomics. PeerJ 4:e2584 doi: 10.7717/peerj.2584 (link)

REPORTING BUGS

Submit suggestions and bug-reports at (link) <https://github.com/torognes/vsearch/issues>, send a pull request on (link) <https://github.com/torognes/vsearch>, or compose a friendly or curmudgeont e-mail to Torbjørn Rognes (link) <torognes@ifi.uio.no>.

AVAILABILITY

Source code and binaries are available at <https://github.com/torognes/vsearch>.

COPYRIGHT

Contact: Torbjørn Rognes <torognes@ifi.uio.no>, Department of Informatics, University of Oslo, PO Box 1080 Blindern, NO-0316 Oslo, Norway

This software is dual-licensed and available under a choice of one of two licenses, either under the terms of the GNU General Public License version 3 or the BSD 2-Clause License.

GNU General Public License version 3

This program is free software: you can redistribute it and/or modify it under the terms of the GNU General Public License as published by the Free Software Foundation, either version 3 of the License, or (at your option) any later version.

This program is distributed in the hope that it will be useful, but WITHOUT ANY WARRANTY; without even the implied warranty of MERCHANTABILITY or FITNESS FOR A PARTICULAR PURPOSE. See the GNU General Public License for more details.

You should have received a copy of the GNU General Public License along with this program. If not, see (link) <http://www.gnu.org/licenses/>.

The BSD 2-Clause License

Redistribution and use in source and binary forms, with or without modification, are permitted provided that the following conditions are met:

1. Redistributions of source code must retain the above copyright notice, this list of conditions and the following disclaimer.

2. Redistributions in binary form must reproduce the above copyright notice, this list of conditions and the following disclaimer in the documentation and/or other materials provided with the distribution.

THIS SOFTWARE IS PROVIDED BY THE COPYRIGHT HOLDERS AND CONTRIBUTORS "AS IS" AND ANY EXPRESS OR IMPLIED WARRANTIES, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE ARE DISCLAIMED. IN NO EVENT SHALL THE COPYRIGHT HOLDER OR CONTRIBUTORS BE LIABLE FOR ANY DIRECT, INDIRECT, INCIDENTAL, SPECIAL, EXEMPLARY, OR CONSEQUENTIAL DAMAGES (INCLUDING, BUT NOT LIMITED TO, PROCUREMENT OF SUBSTITUTE GOODS OR SERVICES; LOSS OF USE, DATA, OR PROFITS; OR BUSINESS INTERRUPTION) HOWEVER CAUSED AND ON ANY THEORY OF LIABILITY, WHETHER IN CONTRACT, STRICT LIABILITY, OR TORT (INCLUDING NEGLIGENCE OR OTHERWISE) ARISING IN ANY WAY OUT OF THE USE OF THIS SOFTWARE, EVEN IF ADVISED OF THE POSSIBILITY OF SUCH DAMAGE.

We would like to thank the authors of the following projects for making their source code available:

	-	vsearch includes code from Google’s CityHash project by Geoff Pike and Jyrki Alakuijala, providing some excellent hash functions available under a MIT license.
	-	vsearch includes code derived from Tatusov and Lipman’s DUST program that is in the public domain.
	-	vsearch includes public domain code written by Alexander Peslyak for the MD5 message digest algorithm.
	-	vsearch includes public domain code written by Steve Reid and others for the SHA1 message digest algorithm.
	-	vsearch binaries may include code from the zlib library, copyright Jean-Loup Gailly and Mark Adler.
	-	vsearch binaries may include code from the bzip2 library, copyright Julian R. Seward.

VERSION HISTORY

New features and important modifications of vsearch (short lived or minor bug releases may not be mentioned):

v1.0.0 released November 28th, 2014

First public release.

v1.0.1 released December 1st, 2014

Bug fixes (sortbysize, semicolon after size annotation in headers) and minor changes (labels as secondary sort key for most sorts, treat T and U as identical for dereplication, only output size in --dbmatched file if --sizeout specified).

v1.0.2 released December 6th, 2014

Bug fixes (ssse3/sse4.1 requirement, memory leak).

v1.0.3 released December 6th, 2014

Bug fix (now writes help to stdout instead of stderr).

v1.0.4 released December 8th, 2014

Added --allpairs_global option. Reduce memory requirements slightly and eliminate memory leaks.

v1.0.5 released December 9th, 2014

Fixes a minor bug with --allpairs_global and --acceptall options.

v1.0.6 released December 14th, 2014

Fixes a memory allocation bug in chimera detection (--uchime_ref option).

v1.0.7 released December 19th, 2014

Fixes a bug in the output from chimera detection with the --uchimeout option.

v1.0.8 released January 22nd, 2015

Introduces several changes and bug fixes:

	-	a new linear memory aligner for alignment of sequences longer than 5,000 nucleotides,
	-	a new --cluster_size command that sorts sequences by decreasing abundance before clustering,
	-	meaning of userfields qlo, qhi, tlo, thi changed for compatibility with usearch,
	-	new userfields qilo, qihi, tilo, tihi give alignment coordinates ignoring terminal gaps,
	-	in --uc output files, a perfect alignment is indicated with a ’=’ sign,
	-	the option --cluster_fast now sorts sequences by decreasing length, then by decreasing abundance and finally by sequence identifier,
	-	default --maxseqlength value set to 50,000 nucleotides,
	-	fix for bug in alignment in rare cases,
	-	fix for lack of detection of under- or overflow in SIMD aligner.

v1.0.9 released January 22nd, 2015

Fixes a bug in the function sorting sequences by decreasing abundance (--sortbysize).

v1.0.10 released January 23rd, 2015

Fixes a bug where the --sizein option was ignored and always treated as on, affecting clustering and dereplication commands.

v1.0.11 released February 5th, 2015

Introduces the possibility to output results in SAM format (for clustering, pairwise alignment and searching).

v1.0.12 released February 6th, 2015

Temporarily fixes a problem with long headers in FASTA files.

v1.0.13 released February 17th, 2015

Fix a memory allocation problem when computing multiple sequence alignments with the --msaout and --consout options, as well as a memory leak. Also increased line buffer for reading FASTA files to 4MB.

v1.0.14 released February 17th, 2015

Fix a bug where the multiple alignment and consensus sequence computed after clustering ignored the strand of the sequences. Also decreased size of line buffer for reading FASTA files to 1MB again due to excessive stack memory usage.

v1.0.15 released February 18th, 2015

Fix bug in calculation of identity metric between sequences when using the MBL definition (--iddef 3).

v1.0.16 released February 19th, 2015

Integrated patches from Debian for increased compatibility with various architectures.

v1.1.0 released February 20th, 2015

Added the --quiet option to suppress all output to stdout and stderr except for warnings and fatal errors. Added the --log option to write messages to a log file.

v1.1.1 released February 20th, 2015

Added info about --log and --quiet options to help text.

v1.1.2 released March 18th, 2015

Fix bug with large datasets. Fix format of help info.

v1.1.3 released March 18th, 2015

Fix more bugs with large datasets.

v1.2.0-1.2.19 released July 6th to September 8th, 2015

Several new commands and options added. Bugs fixed. Documentation updated.

v1.3.0 released September 9th, 2015

Changed to autotools build system.

v1.3.1 released September 14th, 2015

Several new commands and options. Bug fixes.

v1.3.2 released September 15th, 2015

Fixed memory leaks. Added ’-h’ shortcut for help. Removed extra ’v’ in version number.

v1.3.3 released September 15th, 2015

Fixed bug in hexadecimal digits of MD5 and SHA1 digests. Added --samheader option.

v1.3.4 released September 16th, 2015

Fixed compilation problems with zlib and bzip2lib.

v1.3.5 released September 17th, 2015

Minor configuration/makefile changes to compile to native CPU and simplify makefile.

v1.4.0 released September 25th, 2015

Added --sizeorder option.

v1.4.1 released September 29th, 2015

Inserted public domain MD5 and SHA1 code to eliminate dependency on crypto and openssl libraries and their licensing issues.

v1.4.2 released October 2nd, 2015

Dynamic loading of libraries for reading gzip and bzip2 compressed files if available. Circumvention of missing gzoffset function in zlib 1.2.3 and earlier.

v1.4.3 released October 3rd, 2015

Fix a bug with determining amount of memory on some versions of Apple OS X.

v1.4.4 released October 3rd, 2015

Remove debug message.

v1.4.5 released October 6th, 2015

Fix memory allocation bug when reading long FASTA sequences.

v1.4.6 released October 6th, 2015

Fix subtle bug in SIMD alignment code that reduced accuracy.

v1.4.7 released October 7th, 2015

Fixes a problem with searching for or clustering sequences with repeats. In this new version, vsearch looks at all words occurring at least once in the sequences in the initial step. Previously only words occurring exactly once were considered. In addition, vsearch now requires at least 10 words to be shared by the sequences, previously only 6 were required. If the query contains less than 10 words, all words must be present for a match. This change seems to lead to slightly reduced recall, but somewhat increased precision, ending up with slightly improved overall accuracy.

v1.5.0 released October 7th, 2015

This version introduces the new option --minwordmatches that allows the user to specify the minimum number of matching unique words before a sequence is considered further. New default values for different word lengths are also set. The minimum word length is increased to 7.

v1.6.0 released October 9th, 2015

This version adds the relabeling options (--relabel, --relabel_md5 and --relabel_sha1) to the shuffle command. It also adds the --xsize option to the clustering, dereplication, shuffling and sorting commands.

v1.6.1 released October 14th, 2015

Fix bugs and update manual and help text regarding relabelling. Add all relabelling options to the subsampling command. Add the --xsize option to chimera detection, dereplication and fastq filtering commands. Refactoring of code.

v1.7.0 released October 14th, 2015

Add --relabel_keep option.

v1.8.0 released October 19th, 2015

Added --search_exact, --fastx_mask and --fastq_convert commands. Changed most commands to read FASTQ input files as well as FASTA files. Modified --fastx_revcomp and --fastx_subsample to write FASTQ files.

v1.8.1 released November 2nd, 2015

Fixes for compatibility with QIIME and older OS X versions.

v1.9.0 released November 12th, 2015

Added the --fastq_mergepairs command and associated options. This command has not been tested well yet. Included additional files to avoid dependency of autoconf for compilation. Fixed an error where identifiers in fasta headers where not truncated at tabs, just spaces. Fixed a bug in detection of the file format (FASTA/FASTQ) of a gzip compressed input file.

v1.9.1 released November 13th, 2015

Fixed memory leak and a bug in score computation in --fastq_mergepairs, and improved speed.

v1.9.2 released November 17th, 2015

Fixed a bug in the computation of some values with --fastq_stats.

v1.9.3 released November 19th, 2015

Workaround for missing x86intrin.h with old compilers.

v1.9.4 released December 3rd, 2015

Fixed incrementation of counter when relabeling dereplicated sequences.

v1.9.5 released December 3rd, 2015

Fixed bug resulting in inferior chimera detection performance.

v1.9.6 released January 8th, 2016

Fixed bug in aligned sequences produced with --fastapairs and --userout (qrow, trow) options.

v1.9.7 released January 12th, 2016

Masking behavior is changed somewhat to keep the letter case of the input sequences unchanged when no masking is performed. Masking is now performed also during chimera detection. Documentation updated.

v1.9.8 released January 22nd, 2016

Fixed bug causing segfault when chimera detection is performed on extremely short sequences.

v1.9.9 released January 22nd, 2016

Adjusted default minimum number of word matches during searches for improved performance.

v1.9.10 released January 25th, 2016

Fixed bug related to masking and lower case database sequences.

v1.10.0 released February 11th, 2016

Parallelized and improved merging of paired-end reads and adjusted some defaults. Removed progress indicator when stderr is not a terminal. Added --fasta_score option to report chimera scores in FASTA files. Added --rereplicate and --fastq_eestats commands. Fixed typos. Added relabelling to files produced with --consout and --profile options.

v1.10.1 released February 23rd, 2016

Fixed a bug affecting the --fastq_mergepairs command causing FASTQ headers to be truncated at first space (despite the bug fix release 1.9.0 of November 12th, 2015). Full headers are now included in the output (no matter if --notrunclabels is in effect or not).

v1.10.2 released March 18th, 2016

Fixed a bug causing a segmentation fault when running --usearch_global with an empty query sequence. Also fixed a bug causing imperfect alignments to be reported with an alignment string of ’=’ in uc output files. Fixed typos in man file. Fixed fasta/fastq processing code regarding presence or absence of compression library header files.

v1.11.1 released April 13th, 2016

Added strand information in UC file for --derep_fulllength and --derep_prefix. Added expected errors (ee) to header of FASTA files specified with --fastaout and --fastaout_discarded when --eeout or --fastq_eeout option is in effect for fastq_filter and fastq_mergepairs. The options --eeout and --fastq_eeout are now equivalent.

v1.11.2 released June 21st, 2016

Two bugs were fixed. The first issue was related to the --query_cov option that used a different coverage definition than the qcov userfield. The coverage is now defined as the fraction of the whole query sequence length that is aligned with matching or mismatching residues in the target. All gaps are ignored. The other issue was related to the consensus sequences produced during clustering when only N’s were present in some positions. Previously these would be converted to A’s in the consensus. The behaviour is changed so that N’s are produced in the consensus, and it should now be more compatible with usearch.

v2.0.0 released June 24th, 2016

This major new version supports reading from pipes. Two new options are added: --gzip_decompress and --bzip2_decompress. One of these options must be specified if reading compressed input from a pipe, but are not required when reading from ordinary files. The vsearch header that was previously written to stdout is now written to stderr. This enables piping of results for further processing. The file name ’-’ now represent standard input (/dev/stdin) or standard output (/dev/stdout) when reading or writing files, respectively. Code for reading FASTA and FASTQ files has been refactored.

v2.0.1 released June 30th, 2016

Avoid segmentation fault when masking very long sequences.

v2.0.2 released July 5th, 2016

Avoid warnings when compiling with GCC 6.

v2.0.3 released August 2nd, 2016

Fixed bad compiler options resulting in Illegal instruction errors when running precompiled binaries.

v2.0.4 released September 1st, 2016

Improved error message for bad FASTQ quality values. Improved manual.

v2.0.5 released September 9th, 2016

Add options --fastaout_discarded and --fastqout_discarded to output discarded sequences from subsampling to separate files. Updated manual.

v2.1.0 released September 16th, 2016

New command: --fastx_filter. New options: --fastq_maxlen, --fastq_truncee. Allow --minwordmatches down to 3.

v2.1.1 released September 23rd, 2016

Fixed bugs in output to UC-files. Improved help text and manual.

v2.1.2 released September 28th, 2016

Fixed incorrect abundance output from fastx_filter and fastq_filter when relabelling.

v2.2.0 released October 7th, 2016

Added OTU table generation options --biomout, --mothur_shared_out and --otutabout to the clustering and searching commands.

v2.3.0 released October 10th, 2016

Allowed zero-length sequences in FASTA and FASTQ files. Added --fastq_trunclen_keep option. Fixed bug with output of OTU tables to pipes.

v2.3.1 released November 16th, 2016

Fixed bug where --minwordmatches 0 was interpreted as the default minimum word matches for the given word length instead of zero. When used in combination with --maxaccepts 0 and --maxrejects 0 it will allow complete bypass of kmer-based heuristics.

v2.3.2 released November 18th, 2016

Fixed bug where vsearch reported the ordinal number of the target sequence instead of the cluster number in column 2 on H-lines in the uc output file after clustering. For search and alignment commands both usearch and vsearch reports the target sequence number here.

v2.3.3 released December 5th, 2016

A minor speed improvement.

v2.3.4 released December 9th, 2016

Fixed bug in output of sequence profiles and updated documentation.

v2.4.0 released February 8th, 2017

Added support for Linux on Power8 systems (ppc64le) and Windows on x86_64. Improved detection of pipes when reading FASTA and FASTQ files. Corrected option for specifiying output from fastq_eestats command in help text.

v2.4.1 released March 1st, 2017

Fixed an overflow bug in fastq_stats and fastq_eestats affecting analysis of very large FASTQ files. Fixed maximum memory usage reporting on Windows.

v2.4.2 released March 10th, 2017

Default value for fastq_minovlen increased to 16 in accordance with help text and for compatibility with usearch. Minor changes for improved accuracy of paired-end read merging.

v2.4.3 released April 6th, 2017

Fixed bug with progress bar for shuffling. Fixed missing N-lines in UC files with usearch_global, search_exact and allpairs_global when the output_no_hits option was not specified.

v2.4.4 released August 28th, 2017

Fixed a few minor bugs, improved error messages and updated documentation.

v2.5.0 released October 5th, 2017

Support for UDB database files. New commands: fastq_stripright, fastq_eestats2, makeudb_usearch, udb2fasta, udbinfo, and udbstats. New general option: no_progress. New options minsize and maxsize to fastx_filter. Minor bug fixes, error message improvements and documentation updates.

v2.5.1 released October 25th, 2017

Fixed bug with bad default value of 1 instead of 32 for minseqlength when using the makeudb_usearch command.

v2.5.2 released October 30th, 2017

Fixed bug with where ’-’ as an argument to the fastq_eestats2 option was treated literally instead of equivalent to stdin.

v2.6.0 released November 10th, 2017

Rewritten paired-end reads merger with improved accuracy. Decreased default value for fastq_minovlen option from 16 to 10. The default value for the fastq_maxdiffs option is increased from 5 to 10. There are now other more important restrictions that will avoid merging reads that cannot be reliably aligned.

v2.6.1 released December 8th, 2017

Improved parallelisation of paired end reads merging.

v2.6.2 released December 18th, 2017

Fixed option xsize that was partially inactive for commands uchime_denovo, uchime_ref, and fastx_filter.

v2.7.0 released February 13th, 2018

Added commands cluster_unoise, uchime2_denovo and uchime3_denovo contributed by Davide Albanese based on Robert Edgar’s papers. Refactored fasta and fastq print functions as well as code for extraction of abundance and other attributes from the headers.

v2.7.1 released February 16th, 2018

Fix several bugs on Windows related to large files, use of "-" as a file name to mean stdin or stdout, alignment errors, missed kmers and corrupted UDB files. Added documentation of UDB-related commands.

v2.7.2 released April 20th, 2018

Added the sintax command for taxonomic classification. Fixed a bug with incorrect FASTA headers of consensus sequences after clustering.

v2.8.0 released April 24th, 2018

Added the fastq_maxdiffpct option to the fastq_mergepairs command.

v2.8.1 released June 22nd, 2018

Fixes for compilation warnings with GCC 8.

v2.8.2 released August 21st, 2018

Fix for wrong placement of semicolons in header lines in some cases when using the sizeout or xsize options. Reduced memory requirements for full-length dereplication in cases with many duplicate sequences. Improved wording of fastq_mergepairs report. Updated manual regarding use of sizein and sizeout with dereplication. Changed a compiler option.

v2.8.3 released August 31st, 2018

Fix for segmentation fault for --derep_fulllength with --uc.

v2.8.4 released September 3rd, 2018

Further reduce memory requirements for dereplication when not using the uc option. Fix output during subsampling when quiet or log options are in effect.

v2.8.5 released September 26th, 2018

Fixed a bug in fastq_eestats2 that caused the values for large lengths to be much too high when the input sequences had varying lengths.

v2.8.6 released October 9th, 2018

Fixed a bug introduced in version 2.8.2 that caused derep_fulllength to include the full FASTA header in its output instead of stopping at the first space (unless the notrunclabels option is in effect).

v2.9.0 released October 10th, 2018

Added the fastq_join command.

v2.9.1 released October 29th, 2018

Changed compiler options that select the target cpu and tuning to allow the software to run on any 64-bit x86 system, while tuning for more modern variants. Avoid illegal instruction error on some architectures. Update documentation of rereplicate command.

v2.10.0 released December 6th, 2018

Added the sff_convert commmand to convert SFF files to FASTQ. Added some additional option argument checks. Fixed segmentation fault bug after some fatal errors when a log file was specified.

v2.10.1 released December 7th, 2018

Improved sff_convert command. It will now read several variants of the SFF format. It is also able to read from a pipe. Warnings are given if there are minor problems. Errors messages have been improved. Minor speed and memory usage improvements.

v2.10.2 released December 10th, 2018

Fixed bug in sintax with reversed order of domain and kingdom.

v2.10.3 released December 19th, 2018

Ported to Linux on ARMv8 (aarch64). Fixed compilation warning with gcc version 8.1.0 and 8.2.0.

v2.10.4 released January 4th, 2019

Fixed serious bug in x86_64 SIMD alignment code introduced in version 2.10.3. Added link to BioConda in README. Fixed bug in fastq_stats with sequence length 1. Fixed use of equals symbol in UC files for identical sequences with cluster_fast.

v2.11.0 released February 13th, 2019

Added ability to trim and filter paired-end reads using the reverse option with the fastx_filter and fastq_filter commands. Added --xee option to remove ee attributes from FASTA headers. Minor invisible improvement to the progress indicator.

v2.11.1 released February 28th, 2019

Minor change to the handling of the weak_id and id options when using cluster_unoise.

v2.12.0 released March 19th, 2019

Take sequence abundance into account when computing consensus sequences or profiles after clustering. Warn when rereplicating sequences without abundance info. Guess offset 33 in more cases with fastq_chars. Stricter checking of option arguments and option combinations.

v2.13.0 released April 11th, 2019

Added the --fastx_getseq, --fastx_getseqs and --fastx_getsubseq commands to extract sequences from a FASTA or FASTQ file based on their labels. Improved handling of ambiguous nucleotide symbols. Corrected behaviour of --uchime_ref command with and options --self and --selfid. Strict detection of illegal options for each command.

v2.13.1 released April 26th, 2019

Minor changes to the allowed options for each command. All commands now allow the log, quiet and threads options. If more than 1 thread is specified for commands that are not multi-threaded, a warning will be issued. Minor changes to the manual.

v2.13.2 released April 30th, 2019

Fixed bug related to improper handling of newlines on Windows. Allowed option strand plus to uchime_ref for compatibility.

v2.13.3 released April 30th, 2019

Fixed bug in FASTQ parsing introduced in version 2.13.2.

v2.13.4 released May 10th, 2019

Added information about support for gzip- and bzip2-compressed input files to the output of the version command. Adapted source code for compilation on FreeBSD and NetBSD systems.

v2.13.5 released July 2nd, 2019

Added cut command to fragment sequences at restriction sites. Silenced output from the fastq_stats command if quiet option was given. Updated manual.

v2.13.6 released July 2nd, 2019

Added info about cut command to output of help command.

v2.13.7 released September 2nd, 2019

Fixed bug in consensus sequence introduced in version 2.13.0.