bbduk.sh - Filters, trims, or masks reads with kmer matches to an
artifact/contaminant file
bbduk.sh in=<input file> out=<output file>
ref=<contaminant files>
Compares reads to the kmers in a reference dataset, optionally allowing an edit
distance. Splits the reads into two outputs - those that match the reference,
and those that don't. Can also trim (remove) the matching parts of the reads
rather than binning the reads. Please read bbmap/docs/guides/BBDukGuide.txt
for more information.
Input may be stdin or a fasta or fastq file, compressed or uncompressed. If you
pipe via stdin/stdout, please include the file type; e.g. for gzipped fasta
input, set in=stdin.fa.gz
- in=<file>
- Main input. in=stdin.fq will pipe from stdin.
- in2=<file>
- Input for 2nd read of pairs in a different file.
- ref=<file,file>
- Comma-delimited list of reference files.
- In addition to filenames, you may also use the keywords:
adapters, artifacts, phix, lambda, pjet, mtst, kapa
- literal=<seq,seq>
- Comma-delimited list of literal reference sequences.
- touppercase=f
- (tuc) Change all bases upper-case.
- interleaved=auto
- (int) t/f overrides interleaved autodetection.
- qin=auto
- Input quality offset: 33 (Sanger), 64, or auto.
- reads=-1
- If positive, quit after processing X reads or pairs.
- copyundefined=f
- (cu) Process non-AGCT IUPAC reference bases by making all
possible unambiguous copies. Intended for short motifs or adapter
barcodes, as time/memory use is exponential.
- samplerate=1
- Set lower to only process a fraction of input reads.
- samref=<file>
- Optional reference fasta for processing sam files.
- out=<file>
- (outnonmatch) Write reads here that do not contain kmers
matching the database. 'out=stdout.fq' will pipe to standard out.
- out2=<file>
- (outnonmatch2) Use this to write 2nd read of pairs to a
different file.
- outm=<file>
- (outmatch) Write reads here that fail filters. In default
kfilter mode, this means any read with a matching kmer. In any mode, it
also includes reads that fail filters such as minlength, mingc, maxgc,
entropy, etc. In other words, it includes all reads that do not go to
'out'.
- outm2=<file>
- (outmatch2) Use this to write 2nd read of pairs to a
different file.
- outs=<file>
- (outsingle) Use this to write singleton reads whose mate
was trimmed shorter than minlen.
- stats=<file>
- Write statistics about which contamininants were
detected.
- refstats=<file>
- Write statistics on a per-reference-file basis.
- rpkm=<file>
- Write RPKM for each reference sequence (for RNA-seq).
- dump=<file>
- Dump kmer tables to a file, in fasta format.
- duk=<file>
- Write statistics in duk's format. *DEPRECATED*
- nzo=t
- Only write statistics about ref sequences with nonzero
hits.
- overwrite=t
- (ow) Grant permission to overwrite files.
- showspeed=t
- (ss) 'f' suppresses display of processing speed.
- ziplevel=2
- (zl) Compression level; 1 (min) through 9 (max).
- fastawrap=70
- Length of lines in fasta output.
- qout=auto
- Output quality offset: 33 (Sanger), 64, or auto.
- statscolumns=3
- cols) Number of columns for stats output, 3 or 5. 5
includes base counts.
- rename=f
- Rename reads to indicate which sequences they matched.
- refnames=f
- Use names of reference files rather than scaffold IDs.
- trd=f
- Truncate read and ref names at the first whitespace.
- ordered=f
- Set to true to output reads in same order as input.
- maxbasesout=-1
- If positive, quit after writing approximately this many
bases to out (outu/outnonmatch).
- maxbasesoutm=-1
- If positive, quit after writing approximately this many
bases to outm (outmatch).
- json=f
- Print to screen in json format.
- bhist=<file>
- Base composition histogram by position.
- qhist=<file>
- Quality histogram by position.
- qchist=<file>
- Count of bases with each quality value.
- aqhist=<file>
- Histogram of average read quality.
- bqhist=<file>
- Quality histogram designed for box plots.
- lhist=<file>
- Read length histogram.
- phist=<file>
- Polymer length histogram.
- gchist=<file>
- Read GC content histogram.
- ihist=<file>
- Insert size histogram, for paired reads in mapped sam.
- gcbins=100
- Number gchist bins. Set to 'auto' to use read length.
- maxhistlen=6000
- Set an upper bound for histogram lengths; higher uses more
memory. The default is 6000 for some histograms and 80000 for others.
- histbefore=t
- Calculate histograms from reads before processing.
- ehist=<file>
- Errors-per-read histogram.
- qahist=<file>
- Quality accuracy histogram of error rates versus quality
score.
- indelhist=<file>
- Indel length histogram.
- mhist=<file>
- Histogram of match, sub, del, and ins rates by
position.
- idhist=<file>
- Histogram of read count versus percent identity.
- idbins=100
- Number idhist bins. Set to 'auto' to use read length.
- varfile=<file>
- Ignore substitution errors listed in this file when
calculating error rates. Can be generated with CallVariants.
- vcf=<file>
- Ignore substitution errors listed in this VCF file when
calculating error rates.
- ignorevcfindels=t
- Also ignore indels listed in the VCF.
- k=27
- Kmer length used for finding contaminants. Contaminants
shorter than k will not be found. k must be at least 1.
- rcomp=t
- Look for reverse-complements of kmers in addition to
forward kmers.
- maskmiddle=t
- (mm) Treat the middle base of a kmer as a wildcard, to
increase sensitivity in the presence of errors.
- minkmerhits=1
- (mkh) Reads need at least this many matching kmers to be
considered as matching the reference.
- minkmerfraction=0.0
- (mkf) A reads needs at least this fraction of its total
kmers to hit a ref, in order to be considered a match. If this and
minkmerhits are set, the greater is used.
- mincovfraction=0.0
- (mcf) A reads needs at least this fraction of its total
bases to be covered by ref kmers to be considered a match. If specified,
mcf overrides mkh and mkf.
- hammingdistance=0
- (hdist) Maximum Hamming distance for ref kmers (subs only).
Memory use is proportional to (3*K)^hdist.
- qhdist=0
- Hamming distance for query kmers; impacts speed, not
memory.
- editdistance=0
- (edist) Maximum edit distance from ref kmers (subs and
indels).
- Memory use is proportional to (8*K)^edist.
- hammingdistance2=0
- (hdist2) Sets hdist for short kmers, when using mink.
- qhdist2=0
- Sets qhdist for short kmers, when using mink.
- editdistance2=0
- (edist2) Sets edist for short kmers, when using mink.
- forbidn=f
- (fn) Forbids matching of read kmers containing N.
- By default, these will match a reference 'A' if hdist>0
or edist>0, to increase sensitivity.
- removeifeitherbad=t
- (rieb) Paired reads get sent to 'outmatch' if either is
match (or either is trimmed shorter than minlen). Set to false to require
both.
- trimfailures=f
- Instead of discarding failed reads, trim them to 1bp.
- This makes the statistics a bit odd.
- findbestmatch=f
- (fbm) If multiple matches, associate read with sequence
sharing most kmers. Reduces speed.
- skipr1=f
- Don't do kmer-based operations on read 1.
- skipr2=f
- Don't do kmer-based operations on read 2.
- ecco=f
- For overlapping paired reads only. Performs errorcorrection
with BBMerge prior to kmer operations.
- recalibrate=f
- (recal) Recalibrate quality scores. Requires calibration
matrices generated by CalcTrueQuality.
- sam=<file,file>
- If recalibration is desired, and matrices have not already
been generated, BBDuk will create them from the sam file.
- amino=f
- Run in amino acid mode. Some features have not been tested,
but kmer-matching works fine. Maximum k is 12.
- threads=auto
- (t) Set number of threads to use; default is number of
logical processors.
- prealloc=f
- Preallocate memory in table. Allows faster table loading
and more efficient memory usage, for a large reference.
- monitor=f
- Kill this process if it crashes. monitor=600,0.01 would
kill after 600 seconds under 1% usage.
- minrskip=1
- (mns) Force minimal skip interval when indexing reference
kmers.
- 1 means use all, 2 means use every other kmer, etc.
- maxrskip=1
- (mxs) Restrict maximal skip interval when indexing
reference kmers. Normally all are used for scaffolds<100kb, but with
longer scaffolds, up to maxrskip-1 are skipped.
- rskip=
- Set both minrskip and maxrskip to the same value.
- If not set, rskip will vary based on sequence length.
- qskip=1
- Skip query kmers to increase speed. 1 means use all.
- speed=0
- Ignore this fraction of kmer space (0-15 out of 16) in both
reads and reference. Increases speed and reduces memory.
- Note: Do not use more than one of 'speed', 'qskip', and
'rskip'.
Note - if ktrim, kmask, and ksplit are unset, the default behavior is kfilter.
All kmer processing modes are mutually exclusive. Reads only get sent to
'outm' purely based on kmer matches in kfilter mode.
- ktrim=f
- Trim reads to remove bases matching reference kmers.
- Values:
- f (don't trim),
- r (trim to the right),
- l (trim to the left)
- kmask=
- Replace bases matching ref kmers with another symbol.
- Allows any non-whitespace character, and processes
short
- kmers on both ends if mink is set. 'kmask=lc' will convert
masked bases to lowercase.
maskfullycovered=f (mfc) Only mask bases that are fully covered by kmers.
ksplit=f For single-ended reads only. Reads will be split into
- pairs around the kmer.
- If the kmer is at the end of the
- read, it will be trimmed instead.
- Singletons will go to
- out, and pairs will go to outm.
- Do not use ksplit with
- other operations such as quality-trimming or
filtering.
mink=0 Look for shorter kmers at read tips down to this length,
- when k-trimming or masking.
- 0 means disabled. Enabling
- this will disable maskmiddle.
qtrim=f Trim read ends to remove bases with quality below trimq.
- Performed AFTER looking for kmers.
- Values:
- rl (trim both ends),
- f (neither end), r (right end only), l (left end only), w
(sliding window).
- trimq=6
- Regions with average quality BELOW this will be trimmed, if
qtrim is set to something other than f. Can be a floating-point number
like 7.3.
- trimclip=f
- Trim soft-clipped bases from sam files.
- minlength=10
- (ml) Reads shorter than this after trimming will be
discarded. Pairs will be discarded if both are shorter.
- mlf=0
- (minlengthfraction) Reads shorter than this fraction of
original length after trimming will be discarded.
- maxlength=
- Reads longer than this after trimming will be discarded.
Pairs will be discarded only if both are longer.
- minavgquality=0
- (maq) Reads with average quality (after trimming) below
this will be discarded.
- maqb=0
- If positive, calculate maq from this many initial
bases.
- minbasequality=0
- (mbq) Reads with any base below this quality (after
trimming) will be discarded.
- maxns=-1
- If non-negative, reads with more Ns than this (after
trimming) will be discarded.
- mcb=0
- (minconsecutivebases) Discard reads without at least this
many consecutive called bases.
- ottm=f
- (outputtrimmedtomatch) Output reads trimmed to shorter than
minlength to outm rather than discarding.
- tp=0
- (trimpad) Trim this much extra around matching kmers.
- tbo=f
- (trimbyoverlap) Trim adapters based on where paired reads
overlap.
- strictoverlap=t
- Adjust sensitivity for trimbyoverlap mode.
- minoverlap=14
- Require this many bases of overlap for detection.
- mininsert=40
- Require insert size of at least this for overlap. Should be
reduced to 16 for small RNA sequencing.
- tpe=f
- (trimpairsevenly) When kmer right-trimming, trim both reads
to the minimum length of either.
- forcetrimleft=0
- (ftl) If positive, trim bases to the left of this position
(exclusive, 0-based).
- forcetrimright=0
- (ftr) If positive, trim bases to the right of this position
(exclusive, 0-based).
- forcetrimright2=0
- (ftr2) If positive, trim this many bases on the right
end.
- forcetrimmod=0
- (ftm) If positive, right-trim length to be equal to zero,
modulo this number.
- restrictleft=0
- If positive, only look for kmer matches in the leftmost X
bases.
- restrictright=0
- If positive, only look for kmer matches in the rightmost X
bases.
- mingc=0
- Discard reads with GC content below this.
- maxgc=1
- Discard reads with GC content above this.
- gcpairs=t
- Use average GC of paired reads. Also affects gchist.
- tossjunk=f
- Discard reads with invalid characters as bases.
- swift=f
- Trim Swift sequences: Trailing C/T/N R1, leading G/A/N
R2.
- chastityfilter=f
- (cf) Discard reads with id containing ' 1:Y:' or '
2:Y:'.
- barcodefilter=f
- Remove reads with unexpected barcodes if barcodes is set,
or barcodes containing 'N' otherwise. A barcode must be the last part of the
read header.
- Values:
- t: Remove reads with bad barcodes.
- f: Ignore barcodes.
- crash: Crash upon encountering bad barcodes.
- barcodes=
- Comma-delimited list of barcodes or files of barcodes.
- xmin=-1
- If positive, discard reads with a lesser X coordinate.
- ymin=-1
- If positive, discard reads with a lesser Y coordinate.
- xmax=-1
- If positive, discard reads with a greater X
coordinate.
- ymax=-1
- If positive, discard reads with a greater Y
coordinate.
- trimpolya=0
- If greater than 0, trim poly-A or poly-T tails of at least
this length on either end of reads.
- trimpolygleft=0
- If greater than 0, trim poly-G prefixes of at least this
length on the left end of reads. Does not trim poly-C.
- trimpolygright=0
- If greater than 0, trim poly-G tails of at least this
length on the right end of reads. Does not trim poly-C.
- trimpolyg=0
- This sets both left and right at once.
- filterpolyg=0
- If greater than 0, remove reads with a poly-G prefix of at
least this length (on the left).
- Note: there are also equivalent poly-C flags.
- pratio=base,base
- 'pratio=G,C' will print the ratio of G to C polymers.
- plen=20
- Length of homopolymers to count.
- entropy=-1
- Set between 0 and 1 to filter reads with entropy below that
value. Higher is more stringent.
- entropywindow=50
- Calculate entropy using a sliding window of this
length.
- entropyk=5
- Calculate entropy using kmers of this length.
- minbasefrequency=0
- Discard reads with a minimum base frequency below
this.
- entropymask=f
- Values:
- f: Discard low-entropy sequences.
- t: Mask low-entropy parts of sequences with N.
- lc: Change low-entropy parts of sequences to
lowercase.
- entropymark=f
- Mark each base with its entropy value. This is on a scale
of 0-41 and is reported as quality scores, so the output should be fastq
or fasta+qual.
- cardinality=f
- (loglog) Count unique kmers using the LogLog
algorithm.
- cardinalityout=f
- (loglogout) Count unique kmers in output reads.
- loglogk=31
- Use this kmer length for counting.
- loglogbuckets=1999
- Use this many buckets for counting.
- -Xmx
- This will set Java's memory usage, overriding
autodetection.
-
-Xmx20g will specify 20 gigs of RAM, and
-Xmx200m will specify 200 megs. The max is typically 85% of
physical memory.
- -eoom
- This flag will cause the process to exit if an
out-of-memory exception occurs. Requires Java 8u92+.
- -da
- Disable assertions.
Written by Brian Bushnell, Last modified March 21, 2019
Please contact Brian Bushnell at
[email protected] if you encounter any
problems.
This manpage was written by Andreas Tille for the Debian distribution and can be
used for any other usage of the program.