Cutadapt安装
#使用pip安装
$ pip install --user --upgrade cutadapt
#使用conda安装
$ 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
输入输出文件格式
.fasta, .fa, .fna, .fastq, .fq, .gz, .bz2, .xz
多核运行（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 双端测序
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’ (正数)或3’(负数) 固定长度
cutadapt -u 5 -o trimmed.fastq reads.fastq
cutadapt -u -7 -o trimmed.fastq reads.fastq
5 修剪低质量reads
cutadapt -q 10 -o output.fastq input.fastq
cutadapt -q 15,10 -o output.fastq input.fastq
6 修剪reads 3’ 端碱基使得reads到一定长度（10bp）
cutadapt -l 10 -o output.fastq.gz input.fastq.gz
7 Multiple adapters #多种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根据adapter不同输出到不同的文件
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 从一个read修剪多个adapter
cutadapt -g ^ADAPTER -n 5 -o output.fastq.gz input.fastq.gz
常用参数
-g: #剪切reads 5'端adapter(双端测序第一条read)，加$表示adapter锚定在reads 5'端
-a: #剪切reads 3'端adapter(双端测序第一条read)，加$表示adapter锚定在reads3'端
-O MINLENGTH, --overlap=MINLENGTH #adapter与reads最小overlap,才算成功识别; Default: 3
-m LENGTH, --minimum-length=LENGTH: 根据最短长度筛选reads;Default: 0
--discard-untrimmed, --trimmed-only #丢掉不包含adapter的reads
-e ERROR_RATE, --error-rate=ERROR_RATE #adapter匹配允许的最大错配率（错配/匹配片段长度)；Default: 0.1
--no-trim: 不修剪adapter，直接输出满足跳进啊的reads
-u LENGTH, --cut=LENGTH: #修剪reads 5'/3'端碱基,正数：从开始除移除碱基；负数：从末尾处移除碱基；
-q [5'CUTOFF,]3'CUTOFF, --quality-cutoff=[5'CUTOFF,]3'CUTOFF: #修剪低质量碱基
-l LENGTH, --length=LENGTH: #将reads修剪的最终长度
--trim-n: #修剪reads末端的'N'
-o FILE, --output=FILE: #输出文件
--info-file=FILE：每条reads和与reads匹配的adapter的信息
--too-short-output=FILE: #为reads长度最小值设定阈值筛选reads后，要丢弃的部分输出到文件；长度依据m值设定；
--too-long-output=FILE：#为reads长度最大值设定阈值筛选reads后，要丢弃的部分输出到文件；长度依据M值设定；
--untrimmed-output=FILE: #将没有adapter未做修剪的reads输出到一个文件;默认输出到trimmed reads结果文件
--max-n=COUNT：#reads中N的数量，设定整数或小数(N的占比)
双端测序参数
-A ADAPTER： #第二条reads 3'adapter
-G ADAPTER：#第二条reads 5'adapter
-U LENGTH： #从第二条reads上修剪的长度
-p FILE, --paired-output=FILE： #第二条reads的输出结果
--untrimmed-paired-output=FILE：#第一条reads没有adapter时，将第二条reads输出到文件；默认输出到trimmed reads结果文件
参数详解
$ 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$表示congreads 3'端第一个碱基（无法adapter部分匹配识别）
Sequence of an adapter ligated to the 3' end (paired
data: of the first read). The adapter and subsequent
bases are trimmed. If a '$' 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'端第一个碱基匹配adapter（无法adapter部分匹配识别）
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'端都可能出现时使用，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匹配允许的最大错配率（错配/匹配片段长度）
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发生Insertions和deletions
-n COUNT, --times=COUNT #从reads行修剪adapter次数
Remove up to COUNT adapters from each read. Default: 1
-O MINLENGTH, --overlap=MINLENGTH #adapter与reads最小overlap,才算成功识别;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输出reads
--mask-adapter Mask adapters with 'N' characters instead of trimming
them. #识别adapter后，用'N'代替adapter
Additional read modifications:
-u LENGTH, --cut=LENGTH #修剪reads 5'/3'端碱基,正数：从开始除移除碱基；负数：从末尾处移除碱基；
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 #修剪低质量碱基
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 #将reads修剪的最终长度
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，用此参数修改reads的长度
--strip-suffix=STRIP_SUFFIX
Remove this suffix from read names if present. Can be
given multiple times.
-x PREFIX, --prefix=PREFIX #为reads名添加前缀，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 #为reads名添加后缀，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短于最短长度时，丢弃这对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长于最大长度时，丢弃这对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的数量，设定整数或小数(N的占比)
--discard-trimmed, --discard #丢弃只有一个adapter的reads
Discard reads that contain an adapter. Also use -O to
avoid discarding too many randomly matching reads!
--discard-untrimmed, --trimmed-only #丢掉不包含adapter的reads
Discard reads that do not contain the adapter.
Output:
--quiet Print only error messages.
-o FILE, --output=FILE #输出文件
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. #每条reads与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 #为reads长度最大值设定阈值筛选reads后，要丢弃的部分输出到文件；
Write reads that are too short (according to length
specified by -m) to FILE. Default: discard reads
--too-long-output=FILE #将依据最长长度筛选reads后，要丢弃的部分输出到文件；
Write reads that are too long (according to length
specified by -M) to FILE. Default: discard reads
--untrimmed-output=FILE #将没有adapter未做修剪的reads输出到一个文件，而不是输出到trimmed reads结果文件
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: #双端测序参数
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. #第二条reads 3'adapter
-G ADAPTER 5' adapter to be removed from second read in a pair. #第二条reads 5'adapter
-B ADAPTER 5'/3 adapter to be removed from second read in a pair.#第二条reads 5'/3'adapter
-U LENGTH Remove LENGTH bases from second read in a pair #从第二条reads上修剪的长度
-p FILE, --paired-output=FILE #第二条reads的输出结果
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 #第一条reads没有adapter时，将第二条reads输出到文件；默认输出到trimmed reads结果文件
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 #将reads2中太短的reads输出到文件
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 #将reads2中太长的reads输出到文件
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锚定在reads开始处，相当于adapters就是这条reads的前缀一样；
Anchored 5’ adapters的头(a)必须与reads头匹配，才会修剪，除非允许indel；
Anchored 3’ adapters的尾(r)必须与reads尾匹配，才会修剪，除非允许indel；
Regular 3’ adapters修剪
| Reads | Adapter | Reault |
|---|---|---|
| MYSEQUEN | -a ADAPTER | MYSEQUEN |
| MYSEQUENCEADAP | MYSEQUENCE | |
| MYSEQUENCEADAPTER | MYSEQUENCE | |
| MYSEQUENCEADAPTERSOMETHINGELSE | MYSEQUENCE | |
| ADAPTERSOMETHING | 修剪为0bp,导致无意义，reads保留到output |
Regular 5’ adapters修剪**
| Reads | Adapter | Reault |
|---|---|---|
| ADAPTERMYSEQUENCE | -g ADAPTER | MYSEQUENCE |
| DAPTERMYSEQUENCE | MYSEQUENCE | |
| TERMYSEQUENCE | MYSEQUENCE | |
| SOMETHINGADAPTER | 修剪为0bp,导致无意义，reads保留到output |
Anchored 5’ adapters #锚定5’ adapters修剪
| 识别的barcode | Adapter | |
|---|---|---|
| ADAPTER | -g ^ADAPTER | |
| ADAPT | -g ^ADAPTER | |
| ADA | -g ^ADAPTER |
允许错配的话，可能会允许插入的发生：
BADAPTERSOMETHING 也可被-g ^ADAPTER识别，为防止这种现象发生；可以加参数--no-indels
Anchored 3’ adapters #锚定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)
本情况下，5’ 的ADAPTER1是an anchored 5’ adapter
-a ADAPTER1...ADAPTER2: #5’ 正常修剪，3’ 只有 5’存在adapter才会发生修剪；
-a ADAPTER1...ADAPTER2$: #只有 5’和3’ 都存在adapter才会发生修剪；
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’都不是锚定的
-
Both adapters are required. If one of them is not found, the read is not trimmed. #5’和3’同时存在的，adapter才会被剪切
此模式下，可以丢掉不包含adapter的reads；使用参数：
--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
只适用于 (non-anchored) 3’ adapters情况下；
Half matches：只有5’ adapters的情况；
5’ or 3’ adapters
-b ADAPTER (or use the longer spelling --anywhere ADAPTER).
adapter允许出现在reads的任何位置；允许adapter降解；允许partially match;如果adapter前面还有碱基，adapter归于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种情况：mismatches, insertions and deletions
-e ERROR_RATE, --error-rate=ERROR_RATE #adapter匹配允许的最大错配率=错配/匹配片段长度（adapter全长）
Maximum allowed error rate (no. of errors divided by
the length of the matching region). Default: 0.1
--no-indels：禁止adapter发生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允许1bp错配；
40bp adapter，0.1的最大错误率，允许最多错误4bp,这4bp不是均匀分给reads各个位置的；
Multiple adapter occurrences within a single read
5’ and 3’ adapters均识别最左边的adapter
Reducing random matches
-O MINLENGTH, --overlap=MINLENGTH #adapter与reads最小overlap,才算成功识别，最少3bp,再少就丢掉reads
Wildcards
使用N，Y，T这种通配符
cutadapt -a ACGTAANNNNTTAGC -o output.fastq input.fastq
Repeated bases in the adapter sequence
可以去除ploy-A
AAAAAAAAAA)等价于A{10}
cutadapt -a "A{100}" -o output.fastq input.fastq
Modifying reads
对reads的修剪
Removing a fixed number of bases #移除一定数目碱基
-u LENGTH, --cut=LENGTH #修剪reads 5'/3' 碱基，正数表示从 5'移除碱基，负数表示从3'移除碱基
cutadapt -u 5 -o trimmed.fastq reads.fastq
Quality trimming
修剪低质量碱基，默认phred quality + 33；使用64时，需加参数：--quality-base=64
-q [5'CUTOFF,]3'CUTOFF, --quality-cutoff=[5'CUTOFF,]3'CUTOFF
cutadapt -q 10 -o output.fastq input.fastq #默认是3'进行质量修剪
cutadapt -q 15,10 -o output.fastq input.fastq #5'/3' 低质量碱基都修剪
cutadapt -q 15,0 -o output.fastq input.fastq #5'进行质量修剪
Quality trimming of reads using two-color chemistry (NextSeq)
cutadapt --nextseq-trim=20 -o out.fastq input.fastq
Quality trimming algorithm
一段序列质量编码如下：
42, 40, 26, 27, 8, 7, 11, 4, 2, 3
首先减去阈值10
32, 30, 16, 17, -2, -3, 1, -6, -8, -7
从末端开始累加，
(70), (38), 8, -8, -25, -23, -20, -21, -15, -7
因为-25 最小，所以保留-25 之前的碱基， 即保留前4位碱基
Shortening reads to a fixed length
-l LENGTH, --length=LENGTH #修剪reads到固定长度
cutadapt -l 10 -o output.fastq.gz input.fastq.gz
Modifying read names
修改reads名字
-y SUFFIX, --suffix=SUFFIX #为reads名添加后缀，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长度，使用参数--length-tag 'length='更新
Read modification order
Filtering reads
reads的过滤
--minimum-length LENGTH or -m LENGTH #根据最短长度筛选reads;
--too-short-output FILE #为reads长度最小值设定阈值筛选reads后，要丢弃的部分输出到文件；
--maximum-length LENGTH or -M LENGTH #根据最长长度筛选reads;
--too-long-output FILE #为reads长度最大值设定阈值筛选reads后，要丢弃的部分输出到文件；
--untrimmed-output FILE #将没有adapter未做修剪的reads输出到一个文件，而不是输出到trimmed reads结果文件
--discard-trimmed #丢弃只有一个adapter的reads
--discard-trimmed 等价于 --untrimmed-output /dev/null #丢弃没有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: 修剪第二条reads的碱基；
对成对的reads都起作用的参数:
-
-q(along with--quality-base) -
--timesapplies 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
双端测序reads的过滤：
| 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 |
交叉双端测序reads:
也是双端测序，只是所有reads在一个文件；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
正常双端测序，输出这种格式文件：
cutadapt --interleaved -q 20 -a ACGT -A TGCA -o trimmed.1.fastq reads.1.fastq reads.2.fastq
Legacy paired-end read trimming #双端测序修剪
双端测序文件单独处理：（双端测序没有共同的参数的处理）
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
双端测序文件单独处理：（双端测序经过共同参数的处理）
#先处理read1文件，使用-p生成临时文件
cutadapt -q 10 -a ADAPTER_FWD --minimum-length 20 -o tmp.1.fastq -p tmp.2.fastq reads.1.fastq reads.2.fastq
#先处理read1文件，使用临时文件作为输入
cutadapt -q 15 -a ADAPTER_REV --minimum-length 20 -o trimmed.2.fastq -p trimmed.1.fastq tmp.2.fastq tmp.1.fastq
#移除临时文件
rm tmp.1.fastq tmp.2.fastq
Multiple adapters
多条adapter
两条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
-Oparameter) 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: #输出没有adapter的reads到新文件，默认输出到正常trimmed reads结果文件
--untrimmed-paired-output：接受PE测序，没有adapter的reads2输出
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 ##输出没有adapter的reads到文件
PE reads,一般情况下检测reads1的adapter决定这对pair reads;如果想考虑reads2,使用参数-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就停止寻找
#-n COUNT, --times=COUNT #从reads行修剪adapter次数
cutadapt -g ^FIRST -a SECOND -n 2 ...
Illumina TruSeq
单端和双端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.
扩增子测序可以忽略这些警告。
Dealing with N bases
--max-n COUNT #丢掉超过一定数目N 的reads；
--trim-n #修剪reads末端的'N'
Bisulfite sequencing (RRBS)
需要移除3’ 端除adapter外的两个碱基；
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，不允许错配；8-15 bp，允许错配1bp; 16-20 bp允许错配 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. #每条reads与adapter的匹配信息
The alignment algorithm
unit costs
mismatches, insertions and deletions are counted as one error,这样就只需要一个参数(最大错误率)就可以判断是否进行下一步操作
基本理念：在允许的错误率下 ，adapter与reads匹配的区域最大化；
the procedure is as follows:
- Consider all possible overlaps between the two sequences and compute an alignment for each, minimizing the total number of errors in each one.
- Keep only those alignments that do not exceed the specified maximum error rate.
- Then, keep only those alignments that have a maximal number of matches (that is, there is no alignment with more matches).
- If there are multiple alignments with the same number of matches, then keep only those that have the smallest error rate.
- 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.
