cutadapt 使用詳解

Cutadapt安裝

#使用pip安裝
$ pip install --user --upgrade cutadapt
#使用conda安裝
$ conda install -c bioconda cutadapt

基本命令

Usage:
    cutadapt -a ADAPTER options input.fastq
    cutadapt -a AACCGGTT input.fastq > output.fastq
For paired-end reads:
    cutadapt -a ADAPT1 -A ADAPT2 [options] -o out1.fastq -p out2.fastq in1.fastq in2.fastq

輸入輸出文件格式
.fasta, .fa, .fna, .fastq, .fq, .gz, .bz2, .xz
多核運行（Python 3.3 or later）
-j N

例子：

1.1 修剪reads 5'端adapter
cutadapt -g ADAPTER -O 5 -e 0 -o sample.trimmed.fastq sample.fastq --minimum-length 35
--discard-untrimmed --info-file=reads.adapter.txt

1.2 修剪reads 3'端adapter
cutadapt -a ADAPTER -O 5 -e 0 -o sample.trimmed.fastq sample.fastq --minimum-length 35
--discard-untrimmed --info-file=reads.adapter.txt

2 雙端測序
cutadapt -g ADAPTER_FWD -G ADAPTER_REV  -O 5 -e 0 -o sample.trimmed.1.fastq -p sample.trimmed.2.fastq sample.reads_1.fastq sample.reads_2.fastq --minimum-length 35 --discard-untrimmed --info-file=reads.adapter.txt

3 去掉poly-A
cutadapt -a "A{10}" -o output.fastq input.fastq

4 修剪reads 5’ (正數(shù))或3’(負數(shù)) 固定長度
cutadapt -u 5 -o trimmed.fastq reads.fastq
cutadapt -u -7 -o trimmed.fastq reads.fastq

5 修剪低質(zhì)量reads
cutadapt -q 10 -o output.fastq input.fastq
cutadapt -q 15,10 -o output.fastq input.fastq

6 修剪reads 3’ 端堿基使得reads到一定長度（10bp）
cutadapt -l 10 -o output.fastq.gz input.fastq.gz

7 Multiple adapters #多種adapter
cutadapt -g TGAGACACGCA -a AGGCACACAGGG -o output.fastq input.fastq #最佳匹配adapter移除
cutadapt -g file:adapters.fasta -o output.fastq input.fastq

8 Demultiplexing #adapter解析；reads根據(jù)adapter不同輸出到不同的文件
cutadapt -a one=TATA -a two=GCGC -o trimmed-{name}.fastq.gz input.fastq.gz
#result files: demulti-one.fastq.gz, demulti-two.fastq.gz and demulti-unknown.fastq.gz

cutadapt -a file:barcodes.fasta --no-trim --untrimmed-o untrimmed.fastq.gz -o trimmed-{name}.fastq.gz input.fastq.gz
#result files: trimmed-first.1.fastq.gz, trimmed-second.1.fastq.gz, trimmed-unknown.1.fastq.gz and trimmed-first.2.fastq.gz, trimmed-second.2.fastq.gz, trimmed-unknown.2.fastq.gz

9 從一個read修剪多個adapter
cutadapt -g ^ADAPTER -n 5 -o output.fastq.gz input.fastq.gz

常用參數(shù)

-g: #剪切reads 5'端adapter(雙端測序第一條read)，加$表示adapter錨定在reads 5'端
-a: #剪切reads 3'端adapter(雙端測序第一條read)，加$表示adapter錨定在reads3'端
-O MINLENGTH, --overlap=MINLENGTH #adapter與reads最小overlap,才算成功識別; Default: 3
-m LENGTH, --minimum-length=LENGTH: 根據(jù)最短長度篩選reads;Default: 0
--discard-untrimmed, --trimmed-only #丟掉不包含adapter的reads
 -e ERROR_RATE, --error-rate=ERROR_RATE  #adapter匹配允許的最大錯配率（錯配/匹配片段長度)；Default: 0.1
--no-trim: 不修剪adapter，直接輸出滿足跳進啊的reads
-u LENGTH, --cut=LENGTH:  #修剪reads 5'/3'端堿基,正數(shù)：從開始除移除堿基；負數(shù)：從末尾處移除堿基；
-q [5'CUTOFF,]3'CUTOFF, --quality-cutoff=[5'CUTOFF,]3'CUTOFF: #修剪低質(zhì)量堿基
-l LENGTH, --length=LENGTH: #將reads修剪的最終長度
--trim-n: #修剪reads末端的'N'
-o FILE, --output=FILE: #輸出文件
--info-file=FILE：每條reads和與reads匹配的adapter的信息
--too-short-output=FILE: #為reads長度最小值設(shè)定閾值篩選reads后，要丟棄的部分輸出到文件；長度依據(jù)m值設(shè)定；   
--too-long-output=FILE：#為reads長度最大值設(shè)定閾值篩選reads后，要丟棄的部分輸出到文件；長度依據(jù)M值設(shè)定； 
--untrimmed-output=FILE: #將沒有adapter未做修剪的reads輸出到一個文件;默認輸出到trimmed reads結(jié)果文件
--max-n=COUNT：#reads中N的數(shù)量，設(shè)定整數(shù)或小數(shù)(N的占比)

雙端測序參數(shù)
-A ADAPTER：  #第二條reads 3'adapter
-G ADAPTER：#第二條reads 5'adapter
-U LENGTH： #從第二條reads上修剪的長度
-p FILE, --paired-output=FILE： #第二條reads的輸出結(jié)果
--untrimmed-paired-output=FILE：#第一條reads沒有adapter時，將第二條reads輸出到文件；默認輸出到trimmed reads結(jié)果文件

參數(shù)詳解

$ cutadapt --help
Options:
  --version           
  -h, --help            
  --debug               Print debugging information.
  -f FORMAT, --format=FORMAT  #文件格式
                        Input file format：'fasta', 'fastq' or 'sra-fastq'.
                        Ignored when reading csfasta/qual files.
                        Default: auto-detect from file name extension.

  Finding adapters::
    Parameters -a, -g, -b specify adapters to be removed from each read
    (or from the first read in a pair if data is paired). If specified
    multiple times, only the best matching adapter is trimmed (but see the
    --times option). When the special notation 'file:FILE' is used,
    adapter sequences are read from the given FASTA file.

    -a ADAPTER, --adapter=ADAPTER #剪切reads 3'端adapter，加$表示congreads 3'端第一個堿基（無法adapter部分匹配識別）
                        Sequence of an adapter ligated to the 3' end (paired
                        data: of the first read). The adapter and subsequent
                        bases are trimmed. If a '$' character is appended
                        ('anchoring'), the adapter is only found if it is a
                        suffix of the read.匹配adapter
    -g ADAPTER, --front=ADAPTER #剪切reads 5'端adapter,加$表示congreads 5'端第一個堿基匹配adapter（無法adapter部分匹配識別）
                        Sequence of an adapter ligated to the 5' end (paired
                        data: of the first read). The adapter and any
                        preceding bases are trimmed. Partial matches at the 5'
                        end are allowed. If a '^' character is prepended
                        ('anchoring'), the adapter is only found if it is a
                        prefix of the read.
    -b ADAPTER, --anywhere=ADAPTER #adapter在 5'和3'端都可能出現(xiàn)時使用，慎用
                        Sequence of an adapter that may be ligated to the 5'
                        or 3' end (paired data: of the first read). Both types
                        of matches as described under -a und -g are allowed.
                        If the first base of the read is part of the match,
                        the behavior is as with -g, otherwise as with -a. This
                        option is mostly for rescuing failed library preparations
                        -do not use if you know which end your adapter was ligated to!       
    -e ERROR_RATE, --error-rate=ERROR_RATE  #adapter匹配允許的最大錯配率（錯配/匹配片段長度）
                        Maximum allowed error rate (no. of errors divided by
                        the length of the matching region). Default: 0.1
    --no-indels         Allow only mismatches in alignments. Default: allow
                        both mismatches and indels 禁止adapter發(fā)生Insertions和deletions
    -n COUNT, --times=COUNT #從reads行修剪adapter次數(shù)
                        Remove up to COUNT adapters from each read. Default: 1
    -O MINLENGTH, --overlap=MINLENGTH #adapter與reads最小overlap,才算成功識別;Default: 3
                        If the overlap between the read and the adapter is
                        shorter than MINLENGTH, the read is not modified.
                        Reduces the no. of bases trimmed due to random adapter
                        matches. Default: 3
    --match-read-wildcards
                        Interpret IUPAC wildcards in reads. Default: False
    -N, --no-match-adapter-wildcards
                        Do not interpret IUPAC wildcards in adapters.
    --no-trim           Match and redirect reads to output/untrimmed-output as
                        usual, but do not remove adapters. #不修剪adapter輸出reads
    --mask-adapter      Mask adapters with 'N' characters instead of trimming
                        them. #識別adapter后，用'N'代替adapter

  Additional read modifications:
    -u LENGTH, --cut=LENGTH  #修剪reads 5'/3'端堿基,正數(shù)：從開始除移除堿基；負數(shù)：從末尾處移除堿基；
                        Remove bases from each read (first read only if
                        paired). If LENGTH is positive, remove bases from the
                        beginning. If LENGTH is negative, remove bases from
                        the end. Can be used twice if LENGTHs have different
                        signs.
    --nextseq-trim=3'CUTOFF
                        NextSeq-specific quality trimming (each read). Trims
                        also dark cycles appearing as high-quality G bases
                        (EXPERIMENTAL).
    -q [5'CUTOFF,]3'CUTOFF, --quality-cutoff=[5'CUTOFF,]3'CUTOFF #修剪低質(zhì)量堿基
                        Trim low-quality bases from 5' and/or 3' ends of each
                        read before adapter removal. Applied to both reads if
                        data is paired. If one value is given, only the 3' end
                        is trimmed. If two comma-separated cutoffs are given,
                        the 5' end is trimmed with the first cutoff, the 3'
                        end with the second.
    --quality-base=QUALITY_BASE
                        Assume that quality values in FASTQ are encoded as
                        ascii(quality + QUALITY_BASE). This needs to be set to
                        64 for some old Illumina FASTQ files. Default: 33
    -l LENGTH, --length=LENGTH #將reads修剪的最終長度
                        Shorten reads to LENGTH. This and the following
                        modificationsare applied after adapter trimming. 
    --trim-n            Trim N's on ends of reads. #修剪reads末端的'N'
    --length-tag=TAG    Search for TAG followed by a decimal number in the
                        description field of the read. Replace the decimal
                        number with the correct length of the trimmed read.
                        For example, use --length-tag 'length=' to correct
                        fields like 'length=123'. #修剪reads后，用此參數(shù)修改reads的長度
    --strip-suffix=STRIP_SUFFIX
                        Remove this suffix from read names if present. Can be
                        given multiple times.
    -x PREFIX, --prefix=PREFIX #為reads名添加前綴，使用{name}添加adapter名字
                        Add this prefix to read names. Use {name} to insert
                        the name of the matching adapter.
    -y SUFFIX, --suffix=SUFFIX #為reads名添加后綴，使用{name}添加adapter名字
                        Add this suffix to read names; can also include {name}

  Filtering of processed reads:
    -m LENGTH, --minimum-length=LENGTH #修建前后，reads短于最短長度時，丟棄這對reads
                        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 #修建前后，reads長于最大長度時，丟棄這對reads
                        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
     --max-n=COUNT       Discard reads with too many N bases. If COUNT is an
                        integer, it is treated as the absolute number of N
                        bases. If it is between 0 and 1, it is treated as the
                        proportion of N's allowed in a read. #reads中N的數(shù)量，設(shè)定整數(shù)或小數(shù)(N的占比)
    --discard-trimmed, --discard #丟棄只有一個adapter的reads
                        Discard reads that contain an adapter. Also use -O to
                        avoid discarding too many randomly matching reads!
    --discard-untrimmed, --trimmed-only #丟掉不包含adapter的reads
                        Discard reads that do not contain the adapter.  

  Output:
    --quiet             Print only error messages.
    -o FILE, --output=FILE #輸出文件
                        Write trimmed reads to FILE. FASTQ or FASTA format is
                        chosen depending on input. The summary report is sent
                        to standard output. Use '{name}' in FILE to
                        demultiplex reads into multiple files. Default: write
                        to standard output
    --info-file=FILE    Write information about each read and its adapter
                        matches into FILE. #每條reads與adapter的匹配信息
    -r FILE, --rest-file=FILE
                        When the adapter matches in the middle of a read,
                        write the rest (after the adapter) into FILE.
    --wildcard-file=FILE
                        When the adapter has N bases (wildcards), write
                        adapter bases matching wildcard positions to FILE.
                        When there are indels in the alignment, this will
                        often not be accurate.
    --too-short-output=FILE #為reads長度最大值設(shè)定閾值篩選reads后，要丟棄的部分輸出到文件；
                        Write reads that are too short (according to length
                        specified by -m) to FILE. Default: discard reads
    --too-long-output=FILE #將依據(jù)最長長度篩選reads后，要丟棄的部分輸出到文件；
                        Write reads that are too long (according to length
                        specified by -M) to FILE. Default: discard reads
    --untrimmed-output=FILE #將沒有adapter未做修剪的reads輸出到一個文件，而不是輸出到trimmed reads結(jié)果文件
                        Write reads that do not contain any adapter to FILE.
                        Default: output to same file as trimmed reads

  Colorspace options:
    -c, --colorspace    Enable colorspace mode: Also trim the color that is
                        adjacent to the found adapter.
    -d, --double-encode
                        Double-encode colors (map 0,1,2,3,4 to A,C,G,T,N).
    -t, --trim-primer   Trim primer base and the first color (which is the
                        transition to the first nucleotide)
    --strip-f3          Strip the _F3 suffix of read names
    --maq, --bwa        MAQ- and BWA-compatible colorspace output. This
                        enables -c, -d, -t, --strip-f3 and -y '/1'.
    --no-zero-cap       Do not change negative quality values to zero in
                        colorspace data. By default, they are since many tools
                        have problems with negative qualities.
    -z, --zero-cap      Change negative quality values to zero. This is
                        enabled by default when -c/--colorspace is also
                        enabled. Use the above option to disable it.

  Paired-end options:  #雙端測序參數(shù)
    The -A/-G/-B/-U options work like their -a/-b/-g/-u counterparts, but
    are applied to the second read in each pair.

    -A ADAPTER          3' adapter to be removed from second read in a pair. #第二條reads 3'adapter
    -G ADAPTER          5' adapter to be removed from second read in a pair. #第二條reads 5'adapter
    -B ADAPTER          5'/3 adapter to be removed from second read in a pair.#第二條reads 5'/3'adapter
    -U LENGTH           Remove LENGTH bases from second read in a pair #從第二條reads上修剪的長度
    -p FILE, --paired-output=FILE #第二條reads的輸出結(jié)果
                        Write second read in a pair to FILE. 
    --pair-filter=(any|both)
                        Which of the reads in a paired-end read have to match
                        the filtering criterion in order for it to be
                        filtered. Default: any
    --interleaved       Read and write interleaved paired-end reads.
    --untrimmed-paired-output=FILE #第一條reads沒有adapter時，將第二條reads輸出到文件；默認輸出到trimmed reads結(jié)果文件 
                        Write second read in a pair to this FILE when no
                        adapter was found in the first read. Use this option
                        together with --untrimmed-output when trimming paired-
                        end reads. Default: output to same file as trimmed
                        reads
    --too-short-paired-output=FILE #將reads2中太短的reads輸出到文件
                        Write second read in a pair to this file if pair is
                        too short. Use together with --too-short-output.
    --too-long-paired-output=FILE #將reads2中太長的reads輸出到文件
                        Write second read in a pair to this file if pair is
                        too long. Use together with --too-long-output.

Removing adapters

Adapter 移除

Adapter type Command-line option
3’ adapter -a ADAPTER
5’ adapter -g ADAPTER
Anchored 3’ adapter -a ADAPTER$
Anchored 5’ adapter -g ^ADAPTER
5’ or 3’ (both possible) -b ADAPTER
Linked adapter -a ADAPTER1...ADAPTER2
Non-anchored linked adapter -g ADAPTER1...ADAPTER2

Anchored 5’ adapters：adapters錨定在reads開始處，相當(dāng)于adapters就是這條reads的前綴一樣；
Anchored 5’ adapters的頭(a)必須與reads頭匹配，才會修剪，除非允許indel；
Anchored 3’ adapters的尾(r)必須與reads尾匹配，才會修剪，除非允許indel；

Regular 3’ adapters修剪

Reads Adapter Reault
MYSEQUEN -a ADAPTER MYSEQUEN
MYSEQUENCEADAP MYSEQUENCE
MYSEQUENCEADAPTER MYSEQUENCE
MYSEQUENCEADAPTERSOMETHINGELSE MYSEQUENCE
ADAPTERSOMETHING 修剪為0bp,導(dǎo)致無意義，reads保留到output

Regular 5’ adapters修剪**

Reads Adapter Reault
ADAPTERMYSEQUENCE -g ADAPTER MYSEQUENCE
DAPTERMYSEQUENCE MYSEQUENCE
TERMYSEQUENCE MYSEQUENCE
SOMETHINGADAPTER 修剪為0bp,導(dǎo)致無意義，reads保留到output

Anchored 5’ adapters #錨定5’ adapters修剪

識別的barcode Adapter
ADAPTER -g ^ADAPTER
ADAPT -g ^ADAPTER
ADA -g ^ADAPTER

允許錯配的話，可能會允許插入的發(fā)生：

BADAPTERSOMETHING 也可被-g ^ADAPTER識別，為防止這種現(xiàn)象發(fā)生；可以加參數(shù)--no-indels

Anchored 3’ adapters #錨定3’ adapters修剪

reads adapter result
MYSEQUENCEADAP -a ADAPTER$ MYSEQUENCEADAP
MYSEQUENCEADAPTER -a ADAPTER$ MYSEQUENCE
MYSEQUENCEADAPTERSOMETHINGELSE -a ADAPTER$ MYSEQUENCEADAPTERSOMETHINGELSE

Linked adapters (combined 5’ and 3’ adapter)

本情況下，5’ 的ADAPTER1是an anchored 5’ adapter

-a ADAPTER1...ADAPTER2: #5’ 正常修剪，3’ 只有 5’存在adapter才會發(fā)生修剪；

-a ADAPTER1...ADAPTER2$: #只有 5’和3’ 都存在adapter才會發(fā)生修剪；

cutadapt -a FIRST...SECOND -o output.fastq input.fastq
reads adapter result
FIRSTMYSEQUENCESECONDEXTRABASES -a FIRST...SECOND MYSEQUENCE
FIRSTMYSEQUENCESEC MYSEQUENCE
FIRSTMYSEQUE MYSEQUENCE
ANOTHERREADSECOND MYSEQUENCE

Linked adapters without anchoring

-g ADAPTER1...ADAPTER2

  • The 5’ adapter is not anchored by default. (So neither the 5’ nor 3’ adapter are anchored.) #5’和3’都不是錨定的

  • Both adapters are required. If one of them is not found, the read is not trimmed. #5’和3’同時存在的，adapter才會被剪切

    此模式下，可以丟掉不包含adapter的reads；使用參數(shù)：

    --discard-untrimmed, --trimmed-only
    Discard reads that do not contain the adapter.

Linked adapter statistics

=== Adapter 1 ===
Sequence: AAAAAAAAA...TTTTTTTTTT; Type: linked; Length: 9+10; Trimmed: 3 times; Half matches: 2

只適用于 (non-anchored) 3’ adapters情況下；

Half matches：只有5’ adapters的情況；

5’ or 3’ adapters

-b ADAPTER (or use the longer spelling --anywhere ADAPTER).

adapter允許出現(xiàn)在reads的任何位置；允許adapter降解；允許partially match;如果adapter前面還有堿基，adapter歸于3’ adapters

Read before trimming Read after trimming Detected adapter type
MYSEQUENCEADAPTERSOMETHING MYSEQUENCE 3’ adapter
MYSEQUENCEADAPTER MYSEQUENCE 3’ adapter
MYSEQUENCEADAP MYSEQUENCE 3’ adapter
MADAPTER M 3’ adapter
ADAPTERMYSEQUENCE MYSEQUENCE 5’ adapter
PTERMYSEQUENCE MYSEQUENCE 5’ adapter
TERMYSEQUENCE MYSEQUENCE 5’ adapter

Error tolerance

3種情況：mismatches, insertions and deletions

-e ERROR_RATE, --error-rate=ERROR_RATE #adapter匹配允許的最大錯配率=錯配/匹配片段長度（adapter全長）
Maximum allowed error rate (no. of errors divided by
the length of the matching region). Default: 0.1

--no-indels：禁止adapter發(fā)生Insertions和deletions

Sequence: 'LONGADAPTER'; Length: 11; Trimmed: 2 times.

No. of allowed errors:
0-9 bp: 0; 10-11 bp: 1

11bp adapter,前9bp需要完全匹配，10-11 bp允許1bp錯配；

40bp adapter，0.1的最大錯誤率，允許最多錯誤4bp,這4bp不是均勻分給reads各個位置的；

Multiple adapter occurrences within a single read

5’ and 3’ adapters均識別最左邊的adapter

Reducing random matches

-O MINLENGTH, --overlap=MINLENGTH #adapter與reads最小overlap,才算成功識別，最少3bp,再少就丟掉reads

Wildcards

使用N，Y，T這種通配符

cutadapt -a ACGTAANNNNTTAGC -o output.fastq input.fastq

Repeated bases in the adapter sequence

可以去除ploy-A

AAAAAAAAAA)等價于A{10}

cutadapt -a "A{100}" -o output.fastq input.fastq

Modifying reads

對reads的修剪

Removing a fixed number of bases #移除一定數(shù)目堿基

-u LENGTH, --cut=LENGTH #修剪reads 5'/3' 堿基，正數(shù)表示從 5'移除堿基，負數(shù)表示從3'移除堿基

cutadapt -u 5 -o trimmed.fastq reads.fastq

Quality trimming

修剪低質(zhì)量堿基，默認phred quality + 33；使用64時，需加參數(shù)：--quality-base=64

-q [5'CUTOFF,]3'CUTOFF, --quality-cutoff=[5'CUTOFF,]3'CUTOFF

cutadapt -q 10 -o output.fastq input.fastq #默認是3'進行質(zhì)量修剪 

cutadapt -q 15,10 -o output.fastq input.fastq #5'/3' 低質(zhì)量堿基都修剪

cutadapt -q 15,0 -o output.fastq input.fastq #5'進行質(zhì)量修剪

Quality trimming of reads using two-color chemistry (NextSeq)

cutadapt --nextseq-trim=20 -o out.fastq input.fastq

Quality trimming algorithm

一段序列質(zhì)量編碼如下：

42, 40, 26, 27, 8, 7, 11, 4, 2, 3

首先減去閾值10

32, 30, 16, 17, -2, -3, 1, -6, -8, -7

從末端開始累加，

(70), (38), 8, -8, -25, -23, -20, -21, -15, -7

因為-25 最小，所以保留-25 之前的堿基， 即保留前4位堿基

Shortening reads to a fixed length

-l LENGTH, --length=LENGTH #修剪reads到固定長度

cutadapt -l 10 -o output.fastq.gz input.fastq.gz

Modifying read names

修改reads名字

-y SUFFIX, --suffix=SUFFIX #為reads名添加后綴，使用{name}添加adapter名字

cutadapt -a adapter1=ACGT -y ' we found {name}' input.fastq

如果reads名中有reads長度，使用參數(shù)--length-tag 'length='更新

Read modification order

Filtering reads

reads的過濾

--minimum-length LENGTH or -m LENGTH #根據(jù)最短長度篩選reads;

--too-short-output FILE #為reads長度最小值設(shè)定閾值篩選reads后，要丟棄的部分輸出到文件；

--maximum-length LENGTH or -M LENGTH #根據(jù)最長長度篩選reads;

--too-long-output FILE #為reads長度最大值設(shè)定閾值篩選reads后，要丟棄的部分輸出到文件；

--untrimmed-output FILE #將沒有adapter未做修剪的reads輸出到一個文件，而不是輸出到trimmed reads結(jié)果文件

--discard-trimmed #丟棄只有一個adapter的reads

--discard-trimmed 等價于 --untrimmed-output /dev/null #丟棄沒有adapter的reads

Trimming paired-end reads

cutadapt -a ADAPTER_FWD -A ADAPTER_REV -o out.1.fastq -p out.2.fastq reads.1.fastq reads.2.fastq

-U: 修剪第二條reads的堿基；

對成對的reads都起作用的參數(shù):

  • -q (along with --quality-base)
  • --times applies to all the adapters given
  • --no-trim
  • --trim-n
  • --mask
  • --length
  • --length-tag
  • --prefix, --suffix
  • --strip-f3
  • --colorspace, --bwa, -z, --no-zero-cap, --double-encode, --trim-primer

雙端測序reads的過濾：

Filtering option With --pair-filter=any, the pair is discarded if … With -pair-filter=both, the pair is discarded if …
--minimum-length one of the reads is too short both reads are too short
--maximum-length one of the reads is too long both reads are too long
--discard-trimmed one of the reads contains an adapter both reads contain an adapter
--discard-untrimmed one of the reads does not contain an adapter both reads do not contain an adapter
--discard-untrimmed one of the reads does not contain an adapter both reads do not contain an adapter

交叉雙端測序reads:

也是雙端測序，只是所有reads在一個文件；read1在read2前面；

cutadapt --interleaved -q 20 -a ACGT -A TGCA -o trimmed.fastq reads.fastq

cutadapt --interleaved -q 20 -a ACGT -A TGCA -o trimmed.1.fastq -p trimmed.2.fastq reads.fastq

正常雙端測序，輸出這種格式文件：

cutadapt --interleaved -q 20 -a ACGT -A TGCA -o trimmed.1.fastq reads.1.fastq reads.2.fastq

Legacy paired-end read trimming #雙端測序修剪

雙端測序文件單獨處理：（雙端測序沒有共同的參數(shù)的處理）

cutadapt -a ADAPTER_FWD -o trimmed.1.fastq reads1.fastq
cutadapt -a ADAPTER_REV -o trimmed.2.fastq reads2.fastq

cutadapt -q 10 -a ADAPTER_FWD -o trimmed.1.fastq reads1.fastq
cutadapt -q 15 -a ADAPTER_REV -o trimmed.2.fastq reads2.fastq

雙端測序文件單獨處理：（雙端測序經(jīng)過共同參數(shù)的處理）

#先處理read1文件，使用-p生成臨時文件
cutadapt -q 10 -a ADAPTER_FWD --minimum-length 20 -o tmp.1.fastq -p tmp.2.fastq reads.1.fastq reads.2.fastq
#先處理read1文件，使用臨時文件作為輸入
cutadapt -q 15 -a ADAPTER_REV --minimum-length 20 -o trimmed.2.fastq -p trimmed.1.fastq tmp.2.fastq tmp.1.fastq
#移除臨時文件
rm tmp.1.fastq tmp.2.fastq

Multiple adapters

多條adapter

兩條3’ adapters

cutadapt -a TGAGACACGCA -a AGGCACACAGGG -o output.fastq input.fastq

使用adapters文件，fasta格式

cutadapt -a file:adapters.fasta -o output.fastq input.fastq
  • All given adapter sequences are matched to the read.
  • Adapter matches where the overlap length (see the -O parameter) is too small or where the error rate is too high (-e) are removed from further consideration.
  • Among the remaining matches, the one with the greatest number of matching bases is chosen.
  • If there is a tie, the first adapter wins. The order of adapters is the order in which they are given on the command line or in which they are found in the FASTA file.

Named adapters

cutadapt -a My_Adapter=AACCGGTT -o output.fastq input.fastq

Demultiplexing

cutadapt -a one=TATA -a two=GCGC -o trimmed-{name}.fastq.gz input.fastq.gz
#ouput files：demulti-one.fastq.gz, demulti-two.fastq.gz, demulti-unknown.fastq.gz

cutadapt -a file:barcodes.fasta --no-trim --untrimmed-o untrimmed.fastq.gz -o trimmed-{name}.fastq.gz input.fastq.gz

--no-trim: #不修剪reads，保留adapter

--untrimmed-output: #輸出沒有adapter的reads到新文件，默認輸出到正常trimmed reads結(jié)果文件

--untrimmed-paired-output：接受PE測序，沒有adapter的reads2輸出

barcodes.fasta

>barcode01
TTAAGGCC
>barcode02
TAGCTAGC
>barcode03
ATGATGAT

cutadapt -a first=AACCGG -a second=TTTTGG -A ACGTACGT -A TGCATGCA -o trimmed-{name}.1.fastq.gz -p trimmed-{name}.2.fastq.gz input.1.fastq.gz input.2.fastq.gz
#ouput files：
trimmed-first.1.fastq.gz, trimmed-second.1.fastq.gz, trimmed-unknown.1.fastq.gz
trimmed-first.2.fastq.gz, trimmed-second.2.fastq.gz, trimmed-unknown.2.fastq.gz
--untrimmed-paired-output ##輸出沒有adapter的reads到文件
PE reads,一般情況下檢測reads1的adapter決定這對pair reads;如果想考慮reads2,使用參數(shù)-A/-G

Trimming more than one adapter from each read

cutadapt -g ^ADAPTER -n 5 -o output.fastq.gz input.fastq.gz 
#直到在reads中找到5次adapter就停止尋找
#-n COUNT, --times=COUNT #從reads行修剪adapter次數(shù)
cutadapt -g ^FIRST -a SECOND -n 2 ...

Illumina TruSeq

單端和雙端reads1: A + the “TruSeq Indexed Adapter”

cutadapt -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC -o trimmed.fastq.gz reads.fastq.gz

PE reads:

reads2: TruSeq Universal Adapter

cutadapt \
            -a AGATCGGAAGAGCACACGTCTGAACTCCAGTCAC \
            -A AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATT \
            -o trimmed.1.fastq.gz -p trimmed.2.fastq.gz \
            reads.1.fastq.gz reads.2.fastq.gz

Warning about incomplete adapter sequences

WARNING:
    One or more of your adapter sequences may be incomplete.
    Please see the detailed output above.

Bases preceding removed adapters:
  A: 95.5%
  C: 1.0%
  G: 1.6%
  T: 1.6%
  none/other: 0.3%
WARNING:
    The adapter is preceded by "A" extremely often.
    The provided adapter sequence may be incomplete.
    To fix the problem, add "A" to the beginning of the adapter sequence.

擴增子測序可以忽略這些警告。

Dealing with N bases

--max-n COUNT #丟掉超過一定數(shù)目N 的reads；

--trim-n #修剪reads末端的'N'

Bisulfite sequencing (RRBS)

需要移除3’ 端除adapter外的兩個堿基；

cutadapt -a NNADAPTER -o output.fastq input.fastq

Cutadapt’s output

No. of allowed errors:
0-7 bp: 0; 8-15 bp: 1; 16-20 bp: 2

0-7 bp，不允許錯配；8-15 bp，允許錯配1bp; 16-20 bp允許錯配 2bp；

Overview of removed sequences
length  count   expect  max.err error counts
3       140     156.2   0       140
4       57      39.1    0       57
5       50      9.8     0       50
6       35      2.4     0       35
7       13      0.3     0       1 12
8       31      0.1     1       0 31
...
100     397     0.0     3       358 36 3

Format of the info file

--info-file=FILE Write information about each read and its adapter
matches into FILE. #每條reads與adapter的匹配信息

The alignment algorithm

unit costs

mismatches, insertions and deletions are counted as one error,這樣就只需要一個參數(shù)(最大錯誤率)就可以判斷是否進行下一步操作

基本理念：在允許的錯誤率下 ，adapter與reads匹配的區(qū)域最大化；

the procedure is as follows:

  1. Consider all possible overlaps between the two sequences and compute an alignment for each, minimizing the total number of errors in each one.
  2. Keep only those alignments that do not exceed the specified maximum error rate.
  3. Then, keep only those alignments that have a maximal number of matches (that is, there is no alignment with more matches).
  4. If there are multiple alignments with the same number of matches, then keep only those that have the smallest error rate.
  5. If there are still multiple candidates left, choose the alignment that starts at the leftmost position within the read.

In Step 1, the different adapter types are taken into account: Only those overlaps that are actually allowed by the adapter type are actually considered.

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