-

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.

--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.

--bzip2_decompress

When reading from a pipe streaming bzip2-compressed data, decompress the data. This 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 the value to zero to eliminate the wrapping.

--gzip_decompress

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

--label_suffix string

When writing FASTA or FASTQ files, add the suffix string to sequence headers.

--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 information to the log file.

--maxseqlength positive integer

All vsearch operations discard sequences longer than integer (50,000 nucleotides by default).

--minseqlength positive integer

All vsearch operations discard sequences shorter than integer: 1 nucleotide by default for sorting or shuffling, 32 nucleotides for clustering and dereplication as well as the commands --makeudb_usearch, --sintax, and --usearch_global.

--no_progress

Do not show the gradually increasing progress indicator.

--notrunclabels

Do not truncate sequence labels at first space or tab, but use the full header in output files. Turned off by default for all commands except the sintax command.

--quiet

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

--sample string

When writing FASTA or FASTQ files, add the the given sample identifier string to sequence headers. For instance, if the given string is ABC, the text ";sample=ABC" will be added to the header.

--threads positive integer

Number of computation threads to use (1 to 1024). The number of threads should be less than or equal to the number of available CPU cores. The default is to use all available resources and to launch one thread per 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 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 must 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 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. The file name must refer to a FASTA file or to a UDB file. If a UDB file is used, it should be created using the --makeudb_usearch command with the --dbmask dust option.

--dn strictly positive real number

pseudo-count prior on the number of no votes, corresponding to the parameter n in the chimera scoring function (default value is 1.4). Increasing --dn reduces the likelihood of tagging a sequence as a chimera (less false positives, but also more false negatives).

--fasta_score

Add the chimera score to the headers in the fasta output files for chimeras, non-chimeras and borderline sequences, using the format ';uchime_denovo=float;'.

--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_self

Relabel sequences using each sequence itself as a label.

--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.

--sizein

In de novo mode, abundance annotations (pattern '[>;]size=integer[;]') present in sequence headers are taken into account by default (--sizein is always implied). This option is ignored by --uchime_ref.

--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.

--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 strictly positive real number

weight of no votes, corresponding to the parameter beta in the scoring function (default value is 8.0). Increasing --xn reduces the likelihood of tagging a sequence as a chimera (less false positives, but also more false negatives).

--xsize

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

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 <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 identifiers can be generated using the relabelling options (--relabel, --relabel_self, --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 <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 --centroids, --consout and --profile options.

--clusterout_sort

Sort some output files by decreasing abundance instead of input order. It applies to the --consout, --msaout, --profile, --centroids, and --uc options. For --uc, the sorting applies only to the centroid information part (the C lines).

--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:

--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 <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.

--qsegout filename

Write the aligned part of each query sequence to filename in FASTA format.

--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_self

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 ';size=integer;' to sequence headers). If --sizein is 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.

--tsegout filename

Write the aligned part of each target sequence to filename in FASTA format.

--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):

--unoise_alpha real

Specify the alpha parameter to the --cluster_unoise command. The default is 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

VSEARCH can dereplicate sequences with the commands --derep_fulllength, --derep_id, --derep_smallmem, --derep_prefix and --fastx_uniques. The --derep_fulllength command is depreciated and is replaced by the new --fastx_uniques command that can also handle FASTQ files in addition to FASTA files. The --derep_fulllength, --derep_smallmem, and --fastx_uniques commands requires strictly identical sequences of the same length, but ignores upper/lower case and treats T and U as identical symbols. The --derep_id command requires both identical sequences and identical headers/labels. The --derep_prefix command will group sequences with a common prefix and does not require them to be equally long. The --derep_smallmem uses a much smaller amount of memory when dereplicating than the other files, and may be a bit slower and cannot read the input from a pipe. It takes both FASTA and FASTQ files as input but only writes FASTA output to the file specified with the --fastaout option. The --fastx_uniques command can write FASTQ output (specified with --fastqout) or FASTA output (specified with --fastaout) as well as a special tab-separated column text format (with --tabbedout). The other commands can write FASTA output to the file specified with the --output option. All dereplication commands, except --derep_smallmem, can write output to a special UCLUST-like file specified with the --uc option. The --rereplicate command can duplicate sequences in the input file according to the abundance of each input sequence. Other valid options are --fastq_ascii, --fastq_asciiout, --fastq_qmax, --fastq_qmaxout, --fastq_qmin, --fastq_qminout, --fastq_qout_max, --maxuniquesize, --minuniquesize, --relabel, --relabel_keep, --relabel_md5, --relabel_self, --relabel_sha1, --sizein, --sizeout, --strand, --topn, and --xsize.

--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_id filename

Merge strictly identical sequences contained in filename, as with the --derep_fulllength command, but the sequence labels (identifiers) on the header line need to be identical too.

--derep_smallmem filename

Merge strictly identical sequences contained in filename, as with the --derep_fulllength command, but using much less memory. The output is written to a FASTA file specified with the --fastaout option. The output is written in the order that the sequences first appear in the input, and not in decending abundance order as with the other dereplication commands. It can read, but not write FASTQ files. This command cannot read from a pipe, it must be a proper file, as it is read twice. Dereplication is performed with a 128 bit hash function and it is not verified that grouped sequences are identical, however the probability that two different sequences are grouped in a dataset of 1 000 000 000 unique sequences is approximately 1e-21. Multithreading and the options --topn, --uc, or --tabbedout are not supported.

--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.

--fastaout 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 ';size=integer;'. This option is only valid for --fastx_uniques and --derep_smallmem.

--fastqout filename

Write the dereplicated sequences to filename, in fastq 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 fastq header using the pattern ';size=integer;'. This option is only valid for --fastx_uniques.

--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, --sff_convert or --fasta2fastq, 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_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

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

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_qout_max

For --fastx_uniques, indicate that the new quality scores computed when dereplicating FASTQ files should be equal to the maximum (best) of the input quality scores for each position (corresponding to the lowest error probability). The default is to output a quality score corresponding to the average of the error probabilities for each position.

--fastx_uniques filename

Merge strictly identical sequences contained in FASTA or FASTQ file 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. By default, the quality scores in FASTQ output files will correspond to the average error probability of the nucleotides in the each position. If the --fastq_qout_max option is given, the quality score will be the highest (best) quality score observed in each position.

--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 ';size=integer;'. This option is not allowed for --fastx_uniques or --derep_smallmem.

--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_self

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_self, --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 ';size=integer;' to sequence headers). If --sizein is 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.

--tabbedout filename

Output clustering info to the specified tab-separated text file with 6 columns and a row for each input sequence. Column 1 contains the original label/header of the sequence. Column 2 contains the label of the output sequence which is equal to the label/header of the first sequence in each cluster, but potentially relabelled. Column 3 contains the cluster number, starting from 0. Column 4 contains the sequence number within each cluster, starting at 0. Column 5 contains the number of sequences in the cluster. Column 6 contains the original label/header of the first sequence in the cluster before any potential relabelling. This option is only valid for the --fastx_uniques command.

--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):

--xsize

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

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 specified 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

Specify 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

Specify 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

Specify 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

Specify 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.

Analyse, trim, filter, convert, merge, join or reverse complement sequences in FASTA, FASTQ or SFF 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. The --sff_convert command can be used to convert SFF files to FASTQ, while the --fasta2fastq command will convert a FASTA file to a FASTQ file with fake quality scores. Paired-end reads can be merged using the --fastq_mergepairs command or joined with the --fastq_join command. The --fastx_revcomp command will reverse-complements sequences.

--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 output 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.

--fasta2fastq filename

Add a fake nucleotide quality score to the sequences in the given FASTA file and write them to the FASTQ file specified with the --fastqout option. The quality score may be adjusted using the --fastq_qmaxout option (default 41). The --fastq_asciiout option may be used to adjust the FASTQ output quality ASCII base character (default 33).

--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 merging of 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, --sff_convert or --fasta2fastq, 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 an expected error greater than the specified number (value ranging from 0.0 to infinity). For a given sequence, the expected error is the sum of error probabilities for all the positions in the sequence. In practice, the expected error is greater than zero (error probabilities can be small but not null), and at most equal to the length of the sequence (when all positions have an error probability of 1.0).

--fastq_maxee_rate real

When using --fastq_filter or --fastx_filter, discard sequences with an average expected error greater than the specified number (value ranging from 0.0 to 1.0 included). For a given sequence, the average expected error is the sum of error probabilities for all the positions in the sequence, divided by the length of the sequence.

--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. Reads with the same index/position in the forward and reverse files are considered to form a pair, even if their labels are different. Thus, forward and reverse reads must appear in the same order and total number in both files. A warning is emitted if the forward and reverse files contain different numbers of reads. The merged sequences are written 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 (at least 5, 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 minimum and maximum length of the merged sequence may be specified with the --fastq_minmergelen and --fastq_maxmergelen options, respectively. The quality value limits for output files may be specified with the --fastq_qminout and --fastq_qmaxout options, but they apply only to the merged region. Other relevant options are: --fastq_ascii, --fastq_maxee, --fastq_nostagger, --fastq_qmax, --fastq_qmin, 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. Must be at least 5.

--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_mergepairs, --fastq_convert, --sff_convert or --fasta2fastq, specify the maximum quality score used when writing FASTQ files. For the --fasta2fastq command, the value specified here is the fake quality score used for the FASTQ output file. The default is 41, which is usual for recent Sanger/Illumina 1.8+ files. Older formats may use a maximum quality score of 40. The limit only applies to the merged region when using --fastq_mergepairs.

--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_mergepairs, --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. The limit applies only to the merged region when using --fastq_mergepairs.

--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:

--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, --fastx_filter or --fasta2fastq, write to the given FASTQ-formatted file the sequences passing the filter, or the merged or converted 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. When the input is in FASTA format, the following options are not accepted because quality scores are not available: --eeout, --fastq_ascii, --fastq_eeout, --fastq_maxee, --fastq_maxee_rate, --fastq_out, --fastq_qmax, --fastq_qmin, --fastq_truncee, --fastq_truncqual, --fastqout_discarded, --fastqout_discarded_rev, --fastqout_rev.

--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.

--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_self

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.

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):

Results if the --hardmask option is on:

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.

The --orient command can be used to orient the sequences in a given file in either the forward or the reverse complementary direction based on a reference database specified with the --db option. The two strands of each input sequence are compared to the reference database using nucleotide words. If one of the strands share many more words with at least one sequence in the database than the other, that strand is chosen. The correctly oriented sequences may be written to a FASTA file specified with the --fastaout, and to a FASTQ file specified with the --fastqout option (as long as the input was also in FASTA format). If the result is uncertain, because the number of matching words is too similar, the original sequence is written to the file specified with the --notmatched option. The results may also be written to a tab-delimited text file specified with the --tabbedout option. This file will contain the query label, the direction (+, - or ?), the number of matching words on the forward strand, and the number of matching words on the reverse complementary strand. By default, a word length of 12 is used for this command. The word length may be adjusted using the --wordlength option. There has to be at least 4 times as many matches on one strand than the other for a strand to be selected. In addition to the common options, the following options may also be specified for this command: --dbmask, --qmask, --relabel, --relabel_keep, --relabel_md5, --relabel_self, --relabel_sha1, --sizein, and --sizeout.

--db filename

Read the reference database from the given file. It may be in FASTA, FASTQ or UDB format. If an UDB file is used it should have been created with a wordlength of 12.

--fastaout filename

Write the correctly oriented sequences to filename, in fasta format.

--fastqout filename

Write the correctly oriented sequences to filename, in fastq format.

--notmatched filename

Write the sequences with undetermined direction to filename, in the original format.

--orient filename

Orient the sequences in the given file.

--tabbedout filename

Write the resuls to a tab-delimited text file with the specified filename. This file will contain the query label, the direction (+, - or ?), the number of matching words on the forward strand, and the number of matching words on the reverse complementary strand.

The results of the n * (n-1) / 2 pairwise alignments are written to the result files specified with --alnout, --blast6out, --fastapairs --matched, --notmatched, --qsegout, --samout, --tsegout, --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 the fasta sequences contained in filename. Each sequence is compared to all sequencs that come after it in the file, resulting in a total of n * (n-1) / 2 pairwise alignments, where n is the total number of sequences. 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

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.

--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:

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 specified with the --fastaout_discarded_rev option. The relabel options (--relabel, --relabel_self, --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.

--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.

--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:

--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.

--lca_cutoff real

Adjust the fraction of matching hits required for the last common ancestor (LCA) output with the --lcaout option during searches. The default value is 1.0 which requires all hits to match at each taxonomic rank for that rank to be included. If a lower cutoff value is used, e.g. 0.95, a small fraction of non-matching hits are allowed while that rank will still be reported. The argument to this option must be larger than 0.5, but not larger than 1.0.

--lcaout filename

Output last common ancestor (LCA) information about the hits of each query to a text file in a tab-separated format. The first column contains the query id, while the second column contains the taxonomic information. The headers of the sequences in the database must contain taxonomic information in the same format as used with the --sintax command, e.g. "tax=k:Archaea,p:Euryarchaeota,c:Halobacteria". Only the initial parts of the taxonomy that are common to a large fraction of the hits of each query will be output. It is necessary to set the --maxaccepts option to a value different from 1 for this information to be useful. The --top_hits_only option may also be useful. The fraction of matching hits required may be adjusted by the --lca_cutoff option (default 1.0).

--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 non-negative integer

Maximum number of hits to show once the search is terminated (hits are sorted by decreasing identity). Unlimited by default or if the argument it zero. This 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.

--qsegout filename

Write the aligned part of each query sequence to filename in FASTA format.

--query_cov real

Reject if the fraction of the query aligned to the target sequence is lower than real (value ranging from 0.0 to 1.0 included). 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 <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 <https://github.com/samtools/hts-specs> for a complete description of the format):

--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 with an equally high percentage of identity between the query and database sequence sets are written to the output specified with the options --lcaout, --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).

--tsegout filename

Write the aligned part of each target sequence to filename in FASTA format.

--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):

--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.

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_self

Relabel sequences using the sequence itself as the label.

--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_self, --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.

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_self

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 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

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_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_self

Relabel sequences using the sequence itself as the label.

--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.

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

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. The `--randseed` option may be included to specify a seed for initialisation of the random number generator used by the algorithm.

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".

The option --notrunclabels is turned on by default for this command, allowing spaces in the taxonomic identifiers.

--db filename

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

--randseed positive integer

Use integer as seed for the random number generator used in the Sintax algorithm. A given seed always produces the same output order (useful for replicability). Set to 0 to use a pseudo-random seed (default behavior).

--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.

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.

aln

Print a string of M (match/mismatch, i.e. not a gap), 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

The percentage of identity, according to the identity definition specified by the --iddef option. Equal to id0, id1, id2, id3 or id4 below. By default the same as id2.

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.

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 '-'. Empty field if there is no alignment.