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Remove contaminant adapter sequences from your reads prior to other NGS processing

SimilarTo.png: seq_crumbs, FastX_toolkit, PrinSeq, Trimmomatic

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Written by Marcel Martin, cutadapt([1]) will clip or simply filter-out reads that contain a provided linker sequence. It can be tuned to be fault-tolerant and can also be used in reverse-mode to keep only linker-containing reads if this makes sense in your workflow.

Documentation and download link [2]

Download and install

cutadapt (v1.4.1) is a complete command able to find adaptor sequences in short reads and treat them as they diserve (choice of the user). The command line application can be downloaded [3] and was described in a short EMBL publication [4]

Installations and example command to clip adaptors from 'infected' reads, leaving the remaining sequence untouched; Please read the command help for the rich list of options.

# simply install/upgrade with pip if you have it
pip install cutadapt --upgrade
# OR 
# download
cd ${BIOWARE}/download/
wget --no-check-certificate
#decompress it
tar -xzvf cutadapt-1.4.1.tar.gz
# the result is a folder named <cutadapt-1.4.1>
# install the python package
cd cutadapt-1.4.1
python2.7 install
# empty run to get command details
# run example syntax
cutadapt -e ERROR-RATE -a ADAPTER-SEQUENCE input.fastq > output.fastq

command arguments

Usage: cutadapt [options] <FASTA/FASTQ FILE> [<QUALITY FILE>]

Reads a FASTA or FASTQ file, finds and removes adapters,
and writes the changed sequence to standard output.
When finished, statistics are printed to standard error.

Use a dash "-" as file name to read from standard input
(FASTA/FASTQ is autodetected).

If two file names are given, the first must be a .fasta or .csfasta
file and the second must be a .qual file. This is the file format
used by some 454 software and by the SOLiD sequencer.
If you have color space data, you still need to provide the -c option
to correctly deal with color space!

If the name of any input or output file ends with '.gz' or '.bz2', it is
assumed to be gzip-/bzip2-compressed.

If you want to search for the reverse complement of an adapter, you must
provide an additional adapter sequence using another -a, -b or -g parameter.

If the input sequences are in color space, the adapter
can be given in either color space (as a string of digits 0, 1, 2, 3) or in
nucleotide space.


Assuming your sequencing data is available as a FASTQ file, use this
command line:
$ cutadapt -e ERROR-RATE -a ADAPTER-SEQUENCE input.fastq > output.fastq

See the README file for more help and examples.

  --version             show program's version number and exit
  -h, --help            show this help message and exit
  -f FORMAT, --format=FORMAT
                        Input file format; can be either 'fasta', 'fastq' or
                        'sra-fastq'. Ignored when reading csfasta/qual files
                        (default: auto-detect from file name extension).

  Options that influence how the adapters are found:
    Each of the following three parameters (-a, -b, -g) can be used
    multiple times and in any combination to search for an entire set of
    adapters of possibly different types. All of the given adapters will
    be searched for in each read, but only the best matching one will be
    trimmed (but see the --times option).

    -a ADAPTER, --adapter=ADAPTER
                        Sequence of an adapter that was ligated to the 3' end.
                        The adapter itself and anything that follows is
    -b ADAPTER, --anywhere=ADAPTER
                        Sequence of an adapter that was ligated to the 5' or
                        3' end. If the adapter is found within the read or
                        overlapping the 3' end of the read, the behavior is
                        the same as for the -a option. If the adapter overlaps
                        the 5' end (beginning of the read), the initial
                        portion of the read matching the adapter is trimmed,
                        but anything that follows is kept.
    -g ADAPTER, --front=ADAPTER
                        Sequence of an adapter that was ligated to the 5' end.
                        If the adapter sequence starts with the character '^',
                        the adapter is 'anchored'. An anchored adapter must
                        appear in its entirety at the 5' end of the read (it
                        is a prefix of the read). A non-anchored adapter may
                        appear partially at the 5' end, or it may occur within
                        the read. If it is found within a read, the sequence
                        preceding the adapter is also trimmed. In all cases,
                        the adapter itself is trimmed.
    -e ERROR_RATE, --error-rate=ERROR_RATE
                        Maximum allowed error rate (no. of errors divided by
                        the length of the matching region) (default: 0.1)
    --no-indels         Do not allow indels in the alignments, that is, allow
                        only mismatches. This option is currently only
                        supported for anchored 5' adapters ('-g ^ADAPTER')
                        (default: both mismatches and indels are allowed)
    -n COUNT, --times=COUNT
                        Try to remove adapters at most COUNT times. Useful
                        when an adapter gets appended multiple times (default:
    -O LENGTH, --overlap=LENGTH
                        Minimum overlap length. If the overlap between the
                        read and the adapter is shorter than LENGTH, the read
                        is not modified.This reduces the no. of bases trimmed
                        purely due to short random adapter matches (default:
                        Allow 'N's in the read as matches to the adapter
                        (default: False).
    -N, --no-match-adapter-wildcards
                        Do not treat 'N' in the adapter sequence as wildcards.
                        This is needed when you want to search for literal 'N'

  Options for filtering of processed reads:
    --discard-trimmed, --discard
                        Discard reads that contain the adapter instead of
                        trimming them. Also use -O in order to avoid throwing
                        away too many randomly matching reads!
    --discard-untrimmed, --trimmed-only
                        Discard reads that do not contain the adapter.
    -m LENGTH, --minimum-length=LENGTH
                        Discard trimmed reads that are shorter than LENGTH.
                        Reads that are too short even before adapter removal
                        are also discarded. In colorspace, an initial primer
                        is not counted (default: 0).
    -M LENGTH, --maximum-length=LENGTH
                        Discard trimmed reads that are longer than LENGTH.
                        Reads that are too long even before adapter removal
                        are also discarded. In colorspace, an initial primer
                        is not counted (default: no limit).
    --no-trim           Match and redirect reads to output/untrimmed-output as
                        usual, but don't remove the adapters. (default: False.
                        Remove the adapters)

  Options that influence what gets output to where:
    -o FILE, --output=FILE
                        Write the modified sequences to this file instead of
                        standard output and send the summary report to
                        standard output. The format is FASTQ if qualities are
                        available, FASTA otherwise. (default: standard output)
    --info-file=FILE    Write information about each read and its adapter
                        matches into FILE. Currently experimental: Expect the
                        file format to change!
    -r FILE, --rest-file=FILE
                        When the adapter matches in the middle of a read,
                        write the rest (after the adapter) into a file. Use -
                        for standard output.
                        When the adapter has wildcard bases ('N's) write
                        adapter bases matching wildcard positions to FILE. Use
                        - for standard output. When there are indels in the
                        alignment, this may occasionally not be quite
                        Write reads that are too short (according to length
                        specified by -m) to FILE. (default: discard reads)
                        Write reads that are too long (according to length
                        specified by -M) to FILE. (default: discard reads)
                        Write reads that do not contain the adapter to FILE,
                        instead of writing them to the regular output file.
                        (default: output to same file as trimmed)
    -p FILE, --paired-output=FILE
                        Write reads from the paired end input to FILE.

  Additional modifications to the reads:
    -q CUTOFF, --quality-cutoff=CUTOFF
                        Trim low-quality ends from reads before adapter
                        removal. The algorithm is the same as the one used by
                        BWA (Subtract CUTOFF from all qualities; compute
                        partial sums from all indices to the end of the
                        sequence; cut sequence at the index at which the sum
                        is minimal) (default: 0)
                        Assume that quality values are encoded as
                        ascii(quality + QUALITY_BASE). The default (33) is
                        usually correct, except for reads produced by some
                        versions of the Illumina pipeline, where this should
                        be set to 64. (default: 33)
    -x PREFIX, --prefix=PREFIX
                        Add this prefix to read names
    -y SUFFIX, --suffix=SUFFIX
                        Add this suffix to read names
                        Remove this suffix from read names if present. Can be
                        given multiple times.
    -c, --colorspace    Colorspace mode: Also trim the color that is adjacent
                        to the found adapter.
    -d, --double-encode
                        When in color space, double-encode colors (map
                        0,1,2,3,4 to A,C,G,T,N).
    -t, --trim-primer   When in color space, trim primer base and the first
                        color (which is the transition to the first
    --strip-f3          For color space: Strip the _F3 suffix of read names
    --maq, --bwa        MAQ- and BWA-compatible color space output. This
                        enables -c, -d, -t, --strip-f3, -y '/1' and -z.
    --length-tag=TAG    Search for TAG followed by a decimal number in the
                        name of the read (description/comment field of the
                        FASTA or FASTQ file). Replace the decimal number with
                        the correct length of the trimmed read. For example,
                        use --length-tag 'length=' to correct fields like
    -z, --zero-cap      Change negative quality values to zero (workaround to
                        avoid segmentation faults in old BWA versions)

example run

We present here an example command with a bacterial fastq file containing the linker "GATCGGAAGAGCACACGTCTGAACTCCAGTCACACA"

The FastQC report tells us that one specific adaptor is present in 30% of the reads


The next command will filter out reads bearing this adaptor sequence (with 10% error tolerance)

cutadapt -e 0.1 -a GATCGGAAGAGCACACGTCTGAACTCCAGTCACACA SRR576933.fastq > SRR576933-cutadapt.fastq

cutadapt version 1.3
Command line parameters: -e 0.1 -a GATCGGAAGAGCACACGTCTGAACTCCAGTCACACA SRR576933.fastq
Maximum error rate: 10.00%
   No. of adapters: 1
   Processed reads:      3603544
   Processed bases:    129727584 bp (129.7 Mbp)
     Trimmed reads:      1200971 (33.3%)
     Trimmed bases:     41185913 bp (41.2 Mbp) (31.75% of total)
   Too short reads:            0 (0.0% of processed reads)
    Too long reads:            0 (0.0% of processed reads)
        Total time:    139.56 s
     Time per read:      0.039 ms

=== Adapter 1 ===

Adapter 'GATCGGAAGAGCACACGTCTGAACTCCAGTCACACA', length 36, was trimmed 1200971 times.

No. of allowed errors:
0-9 bp: 0; 10-19 bp: 1; 20-29 bp: 2; 30-36 bp: 3

Overview of removed sequences
length  count   expect  max.err error counts
3       45617   56305.4 0       45617
4       10549   14076.3 0       10549
5       2717    3519.1  0       2717
6       655     879.8   0       655
7       146     219.9   0       146
8       39      55.0    0       39
9       100     13.7    0       54 46
10      166     3.4     1       102 64
11      37      0.9     1       14 23
12      31      0.2     1       21 10
13      12      0.1     1       12
14      2       0.0     1       0 2
16      1233    0.0     1       1163 70
17      862     0.0     1       29 833
18      206     0.0     1       197 8 1
19      27      0.0     1       27
20      3       0.0     2       1 2
21      1244    0.0     2       1191 48 5
22      4       0.0     2       4
23      1642    0.0     2       1568 69 5
24      131     0.0     2       124 7
25      27      0.0     2       11 15 1
26      60      0.0     2       54 6
28      6       0.0     2       2 3 1
35      74      0.0     3       63 9 1 1
36      1135381 0.0     3       1060621 67369 6345 1046

cutadapt removed the 1060621 linker-containing reads identified by FastQC, as well as a number of additional imperfect match for a total of 1135381 reads.


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