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一、簡介

Samtools是一個(gè)用于操作sam和bam格式文件的應(yīng)用程序集合，具有眾多的功能。它從SAM（序列比對/映射）格式導(dǎo)入和導(dǎo)出，進(jìn)行排序，合并和索引，并允許快速檢索任何區(qū)域中的讀數(shù)。SAM和BAM格式的比對文件主要由bwa、bowtie2、tophat和hisat2等序列比對工具產(chǎn)生，用于記錄測序reads在參考基因組上的映射信息。其中，BAM格式文件是SAM文件的的二進(jìn)制格式，占據(jù)內(nèi)存較小且運(yùn)算速度更快。

二、基本用法

Samtools由一系列的子命令組成，可以對sam和bam格式的文件進(jìn)行不同的整合與處理。

$ samtools --help

Program: samtools (Tools for alignments in the SAM format)
Version: 1.3.1 (using htslib 1.3.1)

Usage:   samtools <command> [options]

Commands:
  -- Indexing
     dict           create a sequence dictionary file
     faidx          index/extract FASTA
     index          index alignment

  -- Editing
     calmd          recalculate MD/NM tags and '=' bases
     fixmate        fix mate information
     reheader       replace BAM header
     rmdup          remove PCR duplicates
     targetcut      cut fosmid regions (for fosmid pool only)
     addreplacerg   adds or replaces RG tags

  -- File operations
     collate        shuffle and group alignments by name
     cat            concatenate BAMs
     merge          merge sorted alignments
     mpileup        multi-way pileup
     sort           sort alignment file
     split          splits a file by read group
     quickcheck     quickly check if SAM/BAM/CRAM file appears intact
     fastq          converts a BAM to a FASTQ
     fasta          converts a BAM to a FASTA

  -- Statistics
     bedcov         read depth per BED region
     depth          compute the depth
     flagstat       simple stats
     idxstats       BAM index stats
     phase          phase heterozygotes
     stats          generate stats (former bamcheck)

  -- Viewing
     flags          explain BAM flags
     tview          text alignment viewer
     view           SAM<->BAM<->CRAM conversion
     depad          convert padded BAM to unpadded BAM

2.1 構(gòu)建索引(Indexing)

dict -- 對fasta格式的參考基因組序列構(gòu)建字典索引

$ samtools dict

About:   Create a sequence dictionary file from a fasta file
Usage:   samtools dict [options] <file.fa|file.fa.gz>

Options: -a, --assembly STR    assembly
         -H, --no-header       do not print @HD line
         -o, --output STR      file to write out dict file [stdout]
         -s, --species STR     species
         -u, --uri STR         URI [file:///abs/path/to/file.fa]

Example：samtools dict -a GRCh38 -s "Homo sapiens" hg38.fasta -o hg38.dict
構(gòu)建完成后會(huì)生成一個(gè)hg38.dict的參考基因組索引文件

$ head hg38.dict # 查看索引文件的前十行

@HD     VN:1.0  SO:unsorted
@SQ     SN:chr1 LN:248956422    M5:2648ae1bacce4ec4b6cf337dcae37816     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr2 LN:242193529    M5:4bb4f82880a14111eb7327169ffb729b     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr3 LN:198295559    M5:a48af509898d3736ba95dc0912c0b461     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr4 LN:190214555    M5:3210fecf1eb92d5489da4346b3fddc6e     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr5 LN:181538259    M5:f7f05fb7ceea78cbc32ce652c540ff2d     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr6 LN:170805979    M5:6a48dfa97e854e3c6f186c8ff973f7dd     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr7 LN:159345973    M5:94eef2b96fd5a7c8db162c8c74378039     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr8 LN:145138636    M5:c67955b5f7815a9a1edfaa15893d3616     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens
@SQ     SN:chr9 LN:138394717    M5:addd2795560986b7491c40b1faa3978a     UR:file:///data/public/refGenome/bwa_index/hg38/hg38.fa AS:GRCh38  SP:Homo sapiens

faidx -- 對fasta格式的文件建立索引，生成的索引文件以.fai后綴結(jié)尾。

$ samtools faidx

Usage:   samtools faidx <file.fa|file.fa.gz> [<reg> [...]]

Example：samtools faidx hg38.fasta
構(gòu)建完成后會(huì)生成一個(gè)hg38.fasta.fai的索引文件，該文件共有五列，分別表示：

第一列：序列的名稱

第二列：序列的長度

第三列：第一個(gè)堿基的偏移量，從0開始計(jì)數(shù)

第四列：除了最后一行外，序列中每行的堿基數(shù)

第五列：除了最后一行外，序列中每行的長度（包括換行符）

$ head hg38.fasta.fai

chr1    248956422       6       50      51
chr2    242193529       253935563       50      51
chr3    198295559       500972969       50      51
chr4    190214555       703234446       50      51
chr5    181538259       897253299       50      51
chr6    170805979       1082422330      50      51
chr7    159345973       1256644435      50      51
chr8    145138636       1419177334      50      51
chr9    138394717       1567218749      50      51
chr10   133797422       1708381368      50      51

該命令也可以根據(jù)索引文件，快速提取fasta文件中任意區(qū)域的序列文件

# 提取1號(hào)染色體的序列信息
$ samtools faidx ha38.fasta chr1 > hg38.chr1.fa

# 提取1號(hào)染色體200-1000bp之間的序列
$ samtools faidx hg38.fasta chr1:200-1000 > hg38.chr1_200_1000.fa

index -- 對bam格式的比對文件構(gòu)建索引，需對bam文件進(jìn)行排序后才能構(gòu)建索引

$ samtools index

Usage: samtools index [-bc] [-m INT] <in.bam> [out.index]
Options:
  -b       Generate BAI-format index for BAM files [default]
  -c       Generate CSI-format index for BAM files
  -m INT   Set minimum interval size for CSI indices to 2^INT [14]

Example： samtools index aln.sorted.bam
構(gòu)建完成后將產(chǎn)生后綴為.bai的文件，用于快速的隨機(jī)處理

2.2 文件編輯(Editing)

calmd -- recalculate MD/NM tags and '=' bases 計(jì)算MD tag標(biāo)簽值（記錄了mismatch信息）

$ samtools calmd
Usage: samtools calmd [-eubrAES] <aln.bam> <ref.fasta>

Options:
  -e       change identical bases to '='
  -u       uncompressed BAM output (for piping)
  -b       compressed BAM output
  -S       ignored (input format is auto-detected)
  -A       modify the quality string
  -r       compute the BQ tag (without -A) or cap baseQ by BAQ (with -A)
  -E       extended BAQ for better sensitivity but lower specificity
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example：samtools calmd -AEur aln.bam ref.fa

fixmate -- fix mate information 為以名稱排序定位的alignment填入配對坐標(biāo)，ISIZE（inferred insert size推測的插入序列大?。┖团鋵ο鄳?yīng)的標(biāo)簽（flag）

$ samtools fixmate

Usage: samtools fixmate <in.nameSrt.bam> <out.nameSrt.bam>
Options:
  -r           Remove unmapped reads and secondary alignments
  -p           Disable FR proper pair check
  -c           Add template cigar ct tag
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

As elsewhere in samtools, use '-' as the filename for stdin/stdout. The input
file must be grouped by read name (e.g. sorted by name). Coordinated sorted
input is not accepted.

Example：samtools fixmate in.namesorted.sam out.bam

reheader -- replace BAM header 替換bam文件頭注釋信息

$ samtools reheader

Usage: samtools reheader [-P] in.header.sam in.bam > out.bam
   or  samtools reheader [-P] -i in.header.sam file.bam

Options:
    -P, --no-PG      Do not generate an @PG header line.
    -i, --in-place   Modify the bam/cram file directly.
                     (Defaults to outputting to stdout.)

rmdup -- remove PCR duplicates 去除bam文件中的PCR重復(fù)信息

$ samtools rmdup

Usage:  samtools rmdup [-sS] <input.srt.bam> <output.bam>

Option: -s    rmdup for SE reads
        -S    treat PE reads as SE in rmdup (force -s)
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example：samtools rmdup -sS in.sorted.bam output.bam

targetcut -- cut fosmid regions (for fosmid pool only) 此命令僅用于從fosmid混池測序中切除fosmid克隆序列

$ samtools targetcut

Usage: samtools targetcut [-Q minQ] [-i inPen] [-0 em0] [-1 em1] [-2 em2] <in.bam>
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -f, --reference FILE
               Reference sequence FASTA FILE [null]

addreplacerg -- adds or replaces RG tags 該命令用于添加或更改bam文件中的RG tag標(biāo)簽值

$ samtools addreplacerg

Usage: samtools addreplacerg [options] [-r <@RG line> | -R <existing id>] [-o <output.bam>] <input.bam>

Options:
  -m MODE   Set the mode of operation from one of overwrite_all, orphan_only [overwrite_all]
  -o FILE   Where to write output to [stdout]
  -r STRING @RG line text
  -R STRING ID of @RG line in existing header to use
      --input-fmt FORMAT[,OPT[=VAL]]...
               Specify input format (SAM, BAM, CRAM)
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example ：samtools addreplacerg -r 'ID:fish' -r 'LB:1334' -r 'SM:alpha' -o output.bam input.bam

2.3 文件處理(File operations)

collate -- shuffle and group alignments by name 根據(jù)read name信息將bam文件進(jìn)行隨機(jī)打散和分組

$ samtools collate

Usage:   samtools collate [-Ou] [-n nFiles] [-c cLevel] <in.bam> <out.prefix>

Options:
      -O       output to stdout
      -u       uncompressed BAM output
      -l INT   compression level [1]
      -n INT   number of temporary files [64]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools collate -o aln.name_collated.bam aln.sorted.bam

cat -- concatenate BAMs 將多個(gè)bam文件合并為一個(gè)bam文件（與merge命令的區(qū)別是cat不需要將bam文件提前進(jìn)行排序）
```
$ samtools cat

Usage: samtools cat [-h header.sam] [-o out.bam] <in1.bam> [...]
```
- Example: samtools cat -o out.bam in1.bam in2.bam

merge -- merge sorted alignments 將多個(gè)已經(jīng)sort的bam文件進(jìn)行合并

$ samtools merge

Usage: samtools merge [-nurlf] [-h inh.sam] [-b <bamlist.fofn>] <out.bam> <in1.bam> [<in2.bam> ... <inN.bam>]

Options:
  -n         Input files are sorted by read name
  -r         Attach RG tag (inferred from file names)
  -u         Uncompressed BAM output
  -f         Overwrite the output BAM if exist
  -1         Compress level 1
  -l INT     Compression level, from 0 to 9 [-1]
  -R STR     Merge file in the specified region STR [all]
  -h FILE    Copy the header in FILE to <out.bam> [in1.bam]
  -c         Combine @RG headers with colliding IDs [alter IDs to be distinct]
  -p         Combine @PG headers with colliding IDs [alter IDs to be distinct]
  -s VALUE   Override random seed
  -b FILE    List of input BAM filenames, one per line [null]
  -@, --threads INT
             Number of BAM/CRAM compression threads [0]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools merge merged.bam in1.sorted.bam in2.sorted.bam

mpileup -- multi-way pileup 對參考基因組每個(gè)位點(diǎn)做堿基堆積，用于call SNP和INDEL。主要是生成BCF、VCF文件或者pileup一個(gè)或多個(gè)bam文件。比對記錄以在@RG中的樣本名作為區(qū)分標(biāo)識(shí)符。如果樣本標(biāo)識(shí)符缺失，那么每一個(gè)輸入文件則視為一個(gè)樣本

$ samtools mpileup

Usage: samtools mpileup [options] in1.bam [in2.bam [...]]

Input options:
  -6, --illumina1.3+      quality is in the Illumina-1.3+ encoding
  -A, --count-orphans     do not discard anomalous read pairs
  -b, --bam-list FILE     list of input BAM filenames, one per line
  -B, --no-BAQ            disable BAQ (per-Base Alignment Quality)
  -C, --adjust-MQ INT     adjust mapping quality; recommended:50, disable:0 [0]
  -d, --max-depth INT     max per-file depth; avoids excessive memory usage [250]
  -E, --redo-BAQ          recalculate BAQ on the fly, ignore existing BQs
  -f, --fasta-ref FILE    faidx indexed reference sequence file
  -G, --exclude-RG FILE   exclude read groups listed in FILE
  -l, --positions FILE    skip unlisted positions (chr pos) or regions (BED)
  -q, --min-MQ INT        skip alignments with mapQ smaller than INT [0]
  -Q, --min-BQ INT        skip bases with baseQ/BAQ smaller than INT [13]
  -r, --region REG        region in which pileup is generated
  -R, --ignore-RG         ignore RG tags (one BAM = one sample)
  --rf, --incl-flags STR|INT  required flags: skip reads with mask bits unset []
  --ff, --excl-flags STR|INT  filter flags: skip reads with mask bits set
                                            [UNMAP,SECONDARY,QCFAIL,DUP]
  -x, --ignore-overlaps   disable read-pair overlap detection

Output options:
  -o, --output FILE       write output to FILE [standard output]
  -g, --BCF               generate genotype likelihoods in BCF format
  -v, --VCF               generate genotype likelihoods in VCF format

Output options for mpileup format (without -g/-v):
  -O, --output-BP         output base positions on reads
  -s, --output-MQ         output mapping quality

Output options for genotype likelihoods (when -g/-v is used):
  -t, --output-tags LIST  optional tags to output:
               DP,AD,ADF,ADR,SP,INFO/AD,INFO/ADF,INFO/ADR []
  -u, --uncompressed      generate uncompressed VCF/BCF output

SNP/INDEL genotype likelihoods options (effective with -g/-v):
  -e, --ext-prob INT      Phred-scaled gap extension seq error probability [20]
  -F, --gap-frac FLOAT    minimum fraction of gapped reads [0.002]
  -h, --tandem-qual INT   coefficient for homopolymer errors [100]
  -I, --skip-indels       do not perform indel calling
  -L, --max-idepth INT    maximum per-file depth for INDEL calling [250]
  -m, --min-ireads INT    minimum number gapped reads for indel candidates [1]
  -o, --open-prob INT     Phred-scaled gap open seq error probability [40]
  -p, --per-sample-mF     apply -m and -F per-sample for increased sensitivity
  -P, --platforms STR     comma separated list of platforms for indels [all]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Notes: Assuming diploid individuals.

Example: samtools mpileup -C 50 -f ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam
主要參數(shù)解讀：

-A：在檢測變異中，不忽略異常的reads對

-C：用于調(diào)節(jié)比對質(zhì)量的系數(shù)，如果reads中含有過多的錯(cuò)配，不能設(shè)置為零

-D：輸出每個(gè)樣本的reads深度

-l：BED文件或者包含區(qū)域位點(diǎn)的位置列表文件

注意：位置文件包含兩列，染色體和位置，從1開始計(jì)數(shù)。BED文件至少包含3列，染色體、起始和終止位置，開始端從0開始計(jì)數(shù)。

-r：在指定區(qū)域產(chǎn)生pileup，需已建立索引的bam文件，通常和-l參數(shù)一起使用

-o/g/v：輸出文件類型（標(biāo)準(zhǔn)格式文件或者VCF、BCF文件）

-t：設(shè)置FORMAT和INFO的列表內(nèi)容，以逗號(hào)分割

-u：生成未壓縮的VCF和BCF文件

-I：跳過INDEL檢測

-m：候選INDEL的最小間隔的reads

-f：輸入有索引文件的fasta參考序列

-F ：含有間隔reads的最小片段
mpileup生成的結(jié)果包含6列：1) 參考序列名，2) 位置，3) 參考堿基，4) 比對上的reads數(shù)，5) 比對情況，6) 比對上的堿基的質(zhì)量。其中第5列比較復(fù)雜,解釋如下：
a) ‘.’代表與參考序列正鏈匹配。
b) ‘,’代表與參考序列負(fù)鏈匹配。
c) ‘ATCGN’代表在正鏈上的不匹配。
d) ‘a(chǎn)tcgn’代表在負(fù)鏈上的不匹配。
e) ‘*’代表模糊堿基
f) ‘’代表匹配的堿基是一個(gè)read的開始；’'后面緊跟的ascii碼減去33代表比對質(zhì)量；這兩個(gè)符號(hào)修飾的是后面的堿基，其后緊跟的堿基(.,ATCGatcgNn)代表該read的第一個(gè)堿基。
g) ‘$’代表一個(gè)read的結(jié)束，該符號(hào)修飾的是其前面的堿基。
h) 正則式’+[0-9]+[ACGTNacgtn]+’代表在該位點(diǎn)后插入的堿基；
i) 正則式’-[0-9]+[ACGTNacgtn]+’代表在該位點(diǎn)后缺失的堿基；

sort -- sort alignment file 對比對后的bam文件進(jìn)行排序，默認(rèn)按coordinate進(jìn)行排序

$ samtools sort

Usage: samtools sort [options...] [in.bam]
Options:
  -l INT     Set compression level, from 0 (uncompressed) to 9 (best)
  -m INT     Set maximum memory per thread; suffix K/M/G recognized [768M]
  -n         Sort by read name
  -o FILE    Write final output to FILE rather than standard output
  -T PREFIX  Write temporary files to PREFIX.nnnn.bam
  -@, --threads INT
             Set number of sorting and compression threads [1]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools sort -T /tmp/aln.sorted -o aln.sorted.bam aln.bam
主要參數(shù)釋義：

-l：設(shè)置文件壓縮等級，0不壓縮，9壓縮最高

-m：每個(gè)線程運(yùn)行內(nèi)存大?。墒褂肒 M G表示）

-n：按照read名稱進(jìn)行排序

-o：排序后的輸出文件

-T：PREFIX臨時(shí)文件前綴

-@：設(shè)置排序和壓縮的線程數(shù)，默認(rèn)單線程

split -- splits a file by read group 根據(jù)read group標(biāo)簽將bam文件進(jìn)行分割

$ samtools split

Usage: samtools split [-u <unaccounted.bam>[:<unaccounted_header.sam>]]
                      [-f <format_string>] [-v] <merged.bam>
Options:
  -f STRING       output filename format string ["%*_%#.%."]
  -u FILE1        put reads with no RG tag or an unrecognised RG tag in FILE1
  -u FILE1:FILE2  ...and override the header with FILE2
  -v              verbose output
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Format string expansions:
  %%     %
  %*     basename
  %#     @RG index
  %!     @RG ID
  %.     filename extension for output format

Example: samtools split merged.bam

quickcheck -- quickly check if SAM/BAM/CRAM file appears intact 檢查SAM/BAM/CRAM文件的完整性

$ samtools quickcheck

Usage: samtools quickcheck [options] <input> [...]
Options:
  -v              verbose output (repeat for more verbosity)

Notes:

1. In order to use this command effectively, you should check its exit status;
   without any -v options it will NOT print any output, even when some files
   fail the check. One way to use quickcheck might be as a check that all
   BAM files in a directory are okay:

        samtools quickcheck *.bam && echo 'all ok' \
           || echo 'fail!'

   To also determine which files have failed, use the -v option:

        samtools quickcheck -v *.bam > bad_bams.fofn \
           && echo 'all ok' \
           || echo 'some files failed check, see bad_bams.fofn'

Example: samtools quickcheck in1.bam in2.cram

fastq -- converts a BAM to a FASTQ 將bam格式文件轉(zhuǎn)換為fastq文件

$ samtools fastq

Usage: samtools fastq [options...] <in.bam>
Options:
  -0 FILE   write paired reads flagged both or neither READ1 and READ2 to FILE
  -1 FILE   write paired reads flagged READ1 to FILE
  -2 FILE   write paired reads flagged READ2 to FILE
  -f INT    only include reads with all bits set in INT set in FLAG [0]
  -F INT    only include reads with none of the bits set in INT set in FLAG [0]
  -n        don't append /1 and /2 to the read name
  -O        output quality in the OQ tag if present
  -s FILE   write singleton reads to FILE [assume single-end]
  -t        copy RG, BC and QT tags to the FASTQ header line
  -v INT    default quality score if not given in file [1]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools fastq input.bam > output.fastq

fasta -- converts a BAM to a FASTA 將bam格式文件轉(zhuǎn)換為fasta文件

$ samtools fasta

Usage: samtools fasta [options...] <in.bam>
Options:
  -0 FILE   write paired reads flagged both or neither READ1 and READ2 to FILE
  -1 FILE   write paired reads flagged READ1 to FILE
  -2 FILE   write paired reads flagged READ2 to FILE
  -f INT    only include reads with all bits set in INT set in FLAG [0]
  -F INT    only include reads with none of the bits set in INT set in FLAG [0]
  -n        don't append /1 and /2 to the read name
  -s FILE   write singleton reads to FILE [assume single-end]
  -t        copy RG, BC and QT tags to the FASTA header line
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools fasta input.bam > output.fasta

2.4 數(shù)據(jù)統(tǒng)計(jì)(Statistics)

bedcov -- read depth per BED region 統(tǒng)計(jì)給定region區(qū)間內(nèi)reads的深度，每個(gè)堿基的深度疊加在一起

$ samtools bedcov

Usage: samtools bedcov [options] <in.bed> <in1.bam> [...]

  -Q INT       Only count bases of at least INT quality [0]
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools bedcov in.bed aln.sorted.bam

depth -- compute the depth 統(tǒng)計(jì)每個(gè)堿基位點(diǎn)的測序深度，注意計(jì)算前必須對bam文件排序并構(gòu)建索引

$ samtools depth

Usage: samtools depth [options] in1.bam [in2.bam [...]]
Options:
   -a                  output all positions (including zero depth)
   -a -a (or -aa)      output absolutely all positions, including unused ref. sequences
   -b <bed>            list of positions or regions
   -f <list>           list of input BAM filenames, one per line [null]
   -l <int>            read length threshold (ignore reads shorter than <int>)
   -d/-m <int>         maximum coverage depth [8000]
   -q <int>            base quality threshold
   -Q <int>            mapping quality threshold
   -r <chr:from-to>    region
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

The output is a simple tab-separated table with three columns: reference name,
position, and coverage depth.  Note that positions with zero coverage may be
omitted by default; see the -a option.

Example: samtools depth aln.sorted.bam

flagstat -- simple stats 統(tǒng)計(jì)bam文件中read的比對情況
```
$ samtools flagstat

Usage: samtools flagstat [--input-fmt-option OPT=VAL] <in.bam>
```
- Example: samtools flagstat aln.sorted.bam

idxstats -- BAM index stats 統(tǒng)計(jì)bam索引文件里的比對信息
```
$ samtools idxstats

Usage: samtools idxstats <in.bam>
```
- Example: samtools idxstats aln.sorted.bam
- idxstats生成一個(gè)統(tǒng)計(jì)表格，共有4列，分別為”序列名，序列長度，比對上的reads數(shù)，unmapped reads number”。第4列應(yīng)該是paired reads中有一端能匹配到該scaffold上，而另外一端不匹配到任何scaffolds上的reads數(shù)。

phase -- Call and phase heterozygous SNPs

$ samtools phase

Usage:   samtools phase [options] <in.bam>

Options: -k INT    block length [13]
         -b STR    prefix of BAMs to output [null]
         -q INT    min het phred-LOD [37]
         -Q INT    min base quality in het calling [13]
         -D INT    max read depth [256]
         -F        do not attempt to fix chimeras
         -A        drop reads with ambiguous phase

      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools phase test.bam

stats -- generate stats (former bamcheck) 對bam文件做詳細(xì)統(tǒng)計(jì), 統(tǒng)計(jì)結(jié)果可用mics/plot-bamstats作圖

$ samtools stats

About: The program collects statistics from BAM files. The output can be visualized using plot-bamstats.
Usage: samtools stats [OPTIONS] file.bam
       samtools stats [OPTIONS] file.bam chr:from-to
Options:
    -c, --coverage <int>,<int>,<int>    Coverage distribution min,max,step [1,1000,1]
    -d, --remove-dups                   Exclude from statistics reads marked as duplicates
    -f, --required-flag  <str|int>      Required flag, 0 for unset. See also `samtools flags` [0]
    -F, --filtering-flag <str|int>      Filtering flag, 0 for unset. See also `samtools flags` [0]
        --GC-depth <float>              the size of GC-depth bins (decreasing bin size increases memory requirement) [2e4]
    -h, --help                          This help message
    -i, --insert-size <int>             Maximum insert size [8000]
    -I, --id <string>                   Include only listed read group or sample name
    -l, --read-length <int>             Include in the statistics only reads with the given read length []
    -m, --most-inserts <float>          Report only the main part of inserts [0.99]
    -P, --split-prefix <str>            Path or string prefix for filepaths output by -S (default is input filename)
    -q, --trim-quality <int>            The BWA trimming parameter [0]
    -r, --ref-seq <file>                Reference sequence (required for GC-depth and mismatches-per-cycle calculation).
    -s, --sam                           Ignored (input format is auto-detected).
    -S, --split <tag>                   Also write statistics to separate files split by tagged field.
    -t, --target-regions <file>         Do stats in these regions only. Tab-delimited file chr,from,to, 1-based, inclusive.
    -x, --sparse                        Suppress outputting IS rows where there are no insertions.
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools stats aln.sorted.bam
輸出的信息比較多，部分如下：
Summary Numbers，raw total sequences，filtered sequences, reads mapped, reads mapped and paired,reads properly paired等信息
Fragment Qualitites：根據(jù)cycle統(tǒng)計(jì)每個(gè)位點(diǎn)上的堿基質(zhì)量分布
Coverage distribution：深度為1，2，3，，，的堿基數(shù)目
ACGT content per cycle：ACGT在每個(gè)cycle中的比例
Insert sizes：插入長度的統(tǒng)計(jì)
Read lengths：read的長度分布

2.5 可視化(Viewing)

flags -- explain BAM flags 查看不同flag值的含義

$ samtools flags

About: Convert between textual and numeric flag representation
Usage: samtools flags INT|STR[,...]

Flags:
        0x1     PAIRED        .. paired-end (or multiple-segment) sequencing technology
        0x2     PROPER_PAIR   .. each segment properly aligned according to the aligner
        0x4     UNMAP         .. segment unmapped
        0x8     MUNMAP        .. next segment in the template unmapped
        0x10    REVERSE       .. SEQ is reverse complemented
        0x20    MREVERSE      .. SEQ of the next segment in the template is reversed
        0x40    READ1         .. the first segment in the template
        0x80    READ2         .. the last segment in the template
        0x100   SECONDARY     .. secondary alignment
        0x200   QCFAIL        .. not passing quality controls
        0x400   DUP           .. PCR or optical duplicate
        0x800   SUPPLEMENTARY .. supplementary alignment

Example: samtools flags PAIRED,UNMAP,MUNMAP

FLAGS:

0x1	PAIRED	paired-end (or multiple-segment) sequencing technology
0x2	PROPER_PAIR	each segment properly aligned according to the aligner
0x4	UNMAP	segment unmapped
0x8	MUNMAP	next segment in the template unmapped
0x10	REVERSE	SEQ is reverse complemented
0x20	MREVERSE	SEQ of the next segment in the template is reverse complemented
0x40	READ1	the first segment in the template
0x80	READ2	the last segment in the template
0x100	SECONDARY	secondary alignment
0x200	QCFAIL	not passing quality controls
0x400	DUP	PCR or optical duplicate
0x800	SUPPLEMENTARY	supplementary alignment

tview -- text alignment viewer 直觀地顯示reads比對到參考基因組的情況，類似于基因組瀏覽器

$ samtools tview

Usage: samtools tview [options] <aln.bam> [ref.fasta]
Options:
   -d display      output as (H)tml or (C)urses or (T)ext
   -p chr:pos      go directly to this position
   -s STR          display only reads from this sample or group
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --reference FILE
               Reference sequence FASTA FILE [null]

Example: samtools tview aln.sorted.bam ref.fasta
需先對bam文件進(jìn)行排序并構(gòu)建索引

view -- SAM<->BAM<->CRAM conversion 將sam和bam文件進(jìn)行格式互換

$ samtools view

Usage: samtools view [options] <in.bam>|<in.sam>|<in.cram> [region ...]

Options:
  -b       output BAM
  -C       output CRAM (requires -T)
  -1       use fast BAM compression (implies -b)
  -u       uncompressed BAM output (implies -b)
  -h       include header in SAM output
  -H       print SAM header only (no alignments)
  -c       print only the count of matching records
  -o FILE  output file name [stdout]
  -U FILE  output reads not selected by filters to FILE [null]
  -t FILE  FILE listing reference names and lengths (see long help) [null]
  -L FILE  only include reads overlapping this BED FILE [null]
  -r STR   only include reads in read group STR [null]
  -R FILE  only include reads with read group listed in FILE [null]
  -q INT   only include reads with mapping quality >= INT [0]
  -l STR   only include reads in library STR [null]
  -m INT   only include reads with number of CIGAR operations consuming
           query sequence >= INT [0]
  -f INT   only include reads with all bits set in INT set in FLAG [0]
  -F INT   only include reads with none of the bits set in INT set in FLAG [0]
  -x STR   read tag to strip (repeatable) [null]
  -B       collapse the backward CIGAR operation
  -s FLOAT integer part sets seed of random number generator [0];
           rest sets fraction of templates to subsample [no subsampling]
  -@, --threads INT
           number of BAM/CRAM compression threads [0]
  -?       print long help, including note about region specification
  -S       ignored (input format is auto-detected)
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
  -O, --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE
  -T, --reference FILE
               Reference sequence FASTA FILE [null]

Example ：samtools view -bS test.sam > test.bam
重要參數(shù)解讀：

-b：輸出bam格式

-C：輸出CRAM文件

-1：快速壓縮（需要-b）

-u：輸出未壓縮的bam文件，節(jié)約時(shí)間，占據(jù)較多磁盤空間（需要-b）

-h：默認(rèn)輸出sam文件不帶表頭，該參數(shù)設(shè)定后輸出帶表頭信息

-H：僅僅輸出表頭信息

-c：僅打印匹配數(shù)

-o：輸出文件（stdout標(biāo)準(zhǔn)輸出）

-U：輸出沒有經(jīng)過過濾選擇的reads

-t：制表分隔符文件（需要提供額外的參考數(shù)據(jù)，比如參考基因組的索引）

-L：僅包括和bed文件有重疊的reads

-r：僅輸出在STR讀段組中的reads

-R：僅輸出特定reads

-q：定位的質(zhì)量大于INT[默認(rèn)0]

-l：僅輸出在STR 庫中的reads

-F：獲得比對上（mapped）的過濾設(shè)置[默認(rèn)0]

-f：獲得未必對上（unmapped）的過濾設(shè)置[默認(rèn)0]

-T：使用fasta格式的參考序列

depad -- convert padded BAM to unpadded BAM

$ samtools depad

Usage:   samtools depad <in.bam>

Options:
  -s           Output is SAM (default is BAM)
  -S           Input is SAM (default is BAM)
  -u           Uncompressed BAM output (can't use with -s)
  -1           Fast compression BAM output (can't use with -s)
  -T, --reference FILE
               Padded reference sequence file [null]
  -o FILE      Output file name [stdout]
  -?           Longer help
      --input-fmt-option OPT[=VAL]
               Specify a single input file format option in the form
               of OPTION or OPTION=VALUE
      --output-fmt FORMAT[,OPT[=VAL]]...
               Specify output format (SAM, BAM, CRAM)
      --output-fmt-option OPT[=VAL]
               Specify a single output file format option in the form
               of OPTION or OPTION=VALUE

Example: samtools depad input.bam

三、常用示例

sam/bam格式文件互換

$ samtools view -bS -1 test.sam > test.bam # sam轉(zhuǎn)bam
$ samtools view -h test.bam > test.sam # bam轉(zhuǎn)sam

提取比對到參考基因組上的數(shù)據(jù)

$ samtools view -bF 4 test.bam > test.F.bam

提取沒有比對到參考基因組上的數(shù)據(jù)

$ samtools view -bf 4 test.bam > test.f.bam

雙端reads都比對到參考基因組上的數(shù)據(jù)

$ samtools view -bF 12 test.bam > test.12.bam

單端reads1比對到參考基因組上的數(shù)據(jù)

samtools view -bF 4 -f 8  test .bam > test1.bam

單端reads2比對到參考基因組上的數(shù)據(jù)

$ samtools view -bF 8 -f 4 test.bam > test2.bam

提取bam文件中比對到scaffold1上的比對結(jié)果，并保存到sam文件格式

$ samtools view abc.bam scaffold1 > scaffold1.sam

提取scaffold1上能比對到30k到100k區(qū)域的比對結(jié)果

$ samtools view abc.bam scaffold1:30000-100000 > scaffold1_30k-100k.sam

根據(jù)fasta文件，將 header 加入到 sam 或 bam 文件中

$ samtools view -T genome.fasta -h scaffold1.sam > scaffold1.h.sam

call SNP和INDEL等變異信息

$ samtools mpileup -f genome.fasta abc.bam > abc.txt
$ samtools mpileup -gSDf genome.fasta abc.bam > abc.bcf
$ samtools mpileup -guSDf genome.fasta abc.bam | \
           bcftools view -cvNg - > abc.vcf

參考來源：
http://www.htslib.org/doc/samtools.html
https://www.cnblogs.com/xiaofeiIDO/p/6805373.html
https://blog.csdn.net/sunchengquan/article/details/85176940

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Samtools使用大全

Samtools使用大全

一、簡介

二、基本用法

2.1 構(gòu)建索引(Indexing)

2.2 文件編輯(Editing)

2.3 文件處理(File operations)

2.4 數(shù)據(jù)統(tǒng)計(jì)(Statistics)

2.5 可視化(Viewing)

三、常用示例

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Samtools使用大全

一、簡介

二、基本用法

2.1 構(gòu)建索引(Indexing)

2.2 文件編輯(Editing)

2.3 文件處理(File operations)

2.4 數(shù)據(jù)統(tǒng)計(jì)(Statistics)

2.5 可視化(Viewing)

三、常用示例

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一、簡介

二、基本用法

三、常用示例