samtools学习及使用范例,以及官方文档详解

本文章主要参考“菜鸟”的新浪博客，自己只是把自己操作的过程记录下来，供大家参考。

#第一步：把sam文件转换成bam文件,我们得到map.bam文件
system"samtools view -bS map.sam > map.bam";
#第二步：sort 一下 BAM 文件,得到map.sorted.bam
system"samtools sort map.bam map.sorted";
#第三步：创建一个关于bam的索引文件,我们得到一个map.sorted.bam.bai的文件
system"samtools index map.sorted.bam";
#第四步：找snp，这里用的是sort以后的bam文件，如果不是，就会不断的报错
system"samtools mpileup -ugf TAIR10.fas map.sorted.bam | bcftools view -vcg -D100 ->snp.vcf"

总的运行步骤就是上面的四部，我用perl写了一下，这样可以把命令记录下来，当然你需要一次运行一个命令，其他的命令可以先用#给标记成注释，从上往下依次运行一个命令。

如果我们要获取全部的位点的信息，而不是仅仅snp位点，那么我们只需要把最后一行的-v去掉就可以了。

如下：

system"samtools mpileup -ugf TAIR10.fas map.sorted.bam | bcftools view -cg -D100 ->snp.vcf"

再下面有详细的解释-v的作用： Output variant sites only (force -c)：这里有-v这个选项就只输出snp位点，如果没有-v那么就是输出所有的位点（测序所包含的）

上面程序中用到的命令，在下面详细介绍的时候出现，我都会用中文解释

bam是BInary Alignment/Map的简写，Binary就是二进制的意思。和sam的文件具有相同的内容，自然就可以相互转换。

上面是最简单的例子。

我们再来详细的看一看官方的文档。

网址如下：http://samtools.sourceforge.net/samtools.shtml

Manual Reference Pages  -  samtools (1)

NAME

samtools - Utilities for the Sequence Alignment/Map (SAM) format

bcftools - Utilities for the Binary Call Format (BCF) and VCF

CONTENTS

Synopsis
Description
Samtools Commands And Options
Bcftools Commands And Options
Sam Format
Vcf Format
Examples
Limitations
Author
See Also

SYNOPSIS(大纲）：这个大纲其实详细的说明了运行的命令，如果没有特殊要求就可以直接采用了。下面的东西都是针对这个的描述。

samtools view -bt ref_list.txt -o aln.bam aln.sam.gz

samtools sort aln.bam aln.sorted

     ：这个是sort的命令，需要的是aln.bam时你要sort的文件，后面跟的是你可以自己命名的最好和前面保持一致

samtools index aln.sorted.bam

     ：sort以后要用建立一个索引文件就直接用这个命令

samtools idxstats aln.sorted.bam

samtools view aln.sorted.bam chr2:20,100,000-20,200,000

samtools merge out.bam in1.bam in2.bam in3.bam

samtools faidx ref.fasta

samtools pileup -vcf ref.fasta aln.sorted.bam

samtools mpileup -C50 -gf ref.fasta -r chr3:1,000-2,000 in1.bam in2.bam

     ：我们再上面用过的最后snp的提取里

samtools tview aln.sorted.bam ref.fasta

bcftools index in.bcf

bcftools view in.bcf chr2:100-200 > out.vcf

bcftools view -vc in.bcf > out.vcf 2> out.afs

DESCRIPTION

Samtools is a set of utilities that manipulate alignments in the BAM format. It imports from and exports to the SAM (Sequence Alignment/Map) format, does sorting, merging and indexing, and allows to retrieve reads in any regions swiftly.

Samtools is designed to work on a stream. It regards an input file ‘-’ as the standard input (stdin) and an output file ‘-’ as the standard output (stdout). Several commands can thus be combined with Unix pipes. Samtools always output warning and error messages to the standard error output (stderr).

Samtools is also able to open a BAM (not SAM) file on a remote FTP or HTTP server if the BAM file name starts with ‘ftp://’ or ‘http://’. Samtools checks the current working directory for the index file and will download the index upon absence. Samtools does not retrieve the entire alignment file unless it is asked to do so.

SAMTOOLS COMMANDS AND OPTIONS

view	samtools view [-bchuHS] [-t in.refList] [-o output] [-f reqFlag] [-F skipFlag] [-q minMapQ] [-l library] [-r readGroup] [-R rgFile] <in.bam>|<in.sam> [region1 [...]] Extract/print all or sub alignments in SAM or BAM format. If no region is specified, all the alignments will be printed; otherwise only alignments overlapping the specified regions will be output. An alignment may be given multiple times if it is overlapping several regions. A region can be presented, for example, in the following format: ‘chr2’ (the whole chr2), ‘chr2:1000000’ (region starting from 1,000,000bp) or ‘chr2:1,000,000-2,000,000’ (region between 1,000,000 and 2,000,000bp including the end points). The coordinate is 1-based. OPTIONS: -b Output in the BAM format.我们第一步把sam转换成bam的中-bS中-b表示的就是要输出bam的文件 -f INT Only output alignments with all bits in INT present in the FLAG field. INT can be in hex in the format of /^0x[0-9A-F]+/ [0] -F INT Skip alignments with bits present in INT [0] -h Include the header in the output.（再输出文件中包含头文件） -H Output the header only.（只输出头文件） -l STR Only output reads in library STR [null] -o FILE Output file [stdout] -q INT Skip alignments with MAPQ smaller than INT [0] -r STR Only output reads in read group STR [null] -R FILE Output reads in read groups listed in FILE [null] -S Input is in SAM. If @SQ header lines are absent, the ‘-t’ option is required.这里S表示的就是输入的是SAM的格式，如果sam中没有头文件，那么就要用到-t的选项 -c Instead of printing the alignments, only count them and print the total number. All filter options, such as ‘-f’, ‘-F’ and ‘-q’ , are taken into account. -t FILE This file is TAB-delimited. Each line must contain the reference name and the length of the reference, one line for each distinct reference; additional fields are ignored. This file also defines the order of the reference sequences in sorting. If you run ‘samtools faidx <ref.fa>’, the resultant index file <ref.fa>.fai can be used as this <in.ref_list> file. -u Output uncompressed BAM. This option saves time spent on compression/decomprssion and is thus preferred when the output is piped to another samtools command.
tview	samtools tview <in.sorted.bam> [ref.fasta] Text alignment viewer (based on the ncurses library). In the viewer, press ‘?’ for help and press ‘g’ to check the alignment start from a region in the format like ‘chr10:10,000,000’ or ‘=10,000,000’ when viewing the same reference sequence. 这个命令是查看的命令，看到的是map以后覆盖度的文件，samtools tview .bam文件 .ref文件
mpileup	samtools mpileup [-EBug] [-C capQcoef] [-r reg] [-f in.fa] [-l list] [-M capMapQ][-Q minBaseQ] [-q minMapQ] in.bam [in2.bam [...]] Generate BCF or pileup for one or multiple BAM files. Alignment records are grouped by sample identifiers in @RG header lines. If sample identifiers are absent, each input file is regarded as one sample. In the pileup format (without -uor-g), each line represents a genomic position, consisting of chromosome name, coordinate, reference base, read bases, read qualities and alignment mapping qualities. Information on match, mismatch, indel, strand, mapping quality and start and end of a read are all encoded at the read base column. At this column, a dot stands for a match to the reference base on the forward strand, a comma for a match on the reverse strand, a ’>’ or ’<’ for a reference skip, ‘ACGTN’ for a mismatch on the forward strand and ‘acgtn’ for a mismatch on the reverse strand. A pattern ‘\+[0-9]+[ACGTNacgtn]+’ indicates there is an insertion between this reference position and the next reference position. The length of the insertion is given by the integer in the pattern, followed by the inserted sequence. Similarly, a pattern ‘-[0-9]+[ACGTNacgtn]+’ represents a deletion from the reference. The deleted bases will be presented as ‘*’ in the following lines. Also at the read base column, a symbol ‘^’ marks the start of a read. The ASCII of the character following ‘^’ minus 33 gives the mapping quality. A symbol ‘$’ marks the end of a read segment. Input Options: -6 Assume the quality is in the Illumina 1.3+ encoding. -A Do not skip anomalous read pairs in variant calling. -B Disable probabilistic realignment for the computation of base alignment quality (BAQ). BAQ is the Phred-scaled probability of a read base being misaligned. Applying this option greatly helps to reduce false SNPs caused by misalignments. -b FILE List of input BAM files, one file per line [null] -C INT Coefficient for downgrading mapping quality for reads containing excessive mismatches. Given a read with a phred-scaled probability q of being generated from the mapped position, the new mapping quality is about sqrt((INT-q)/INT)*INT. A zero value disables this functionality; if enabled, the recommended value for BWA is 50. [0] -d INT At a position, read maximally INT reads per input BAM. [250] -E Extended BAQ computation. This option helps sensitivity especially for MNPs, but may hurt specificity a little bit. -f FILE The faidx-indexed reference file in the FASTA format. The file can be optionally compressed by razip. [null]：要有一个参考序列 -l FILE BED or position list file containing a list of regions or sites where pileup or BCF should be generated [null] -q INT Minimum mapping quality for an alignment to be used [0] -Q INT Minimum base quality for a base to be considered [13] -r STR Only generate pileup in region STR [all sites] Output Options:输出选项   -D Output per-sample read depth 读取的深度，可以设定值比如-D100 -g Compute genotype likelihoods and output them in the binary call format (BCF). -S Output per-sample Phred-scaled strand bias P-value -u Similar to -g except that the output is uncompressed（未压缩的） BCF, which is preferred for piping. Options for Genotype Likelihood Computation (for -g or -u):   -e INT Phred-scaled gap extension sequencing error probability. Reducing INTleads to longer indels. [20] -h INT Coefficient for modeling homopolymer errors. Given an l-long homopolymer run, the sequencing error of an indel of size s is modeled as INT*s/l. [100] -I Do not perform INDEL calling -L INT Skip INDEL calling if the average per-sample depth is above INT. [250] -o INT Phred-scaled gap open sequencing error probability. Reducing INT leads to more indel calls. [40] -P STR Comma dilimited list of platforms (determined by @RG-PL) from which indel candidates are obtained. It is recommended to collect indel candidates from sequencing technologies that have low indel error rate such as ILLUMINA. [all]
reheader	samtools reheader <in.header.sam> <in.bam> Replace the header in in.bam with the header in in.header.sam. This command is much faster than replacing the header with a BAM->SAM->BAM conversion.
cat	samtools cat [-h header.sam] [-o out.bam] <in1.bam> <in2.bam> [ ... ] Concatenate BAMs. The sequence dictionary of each input BAM must be identical, although this command does not check this. This command uses a similar trick toreheader which enables fast BAM concatenation.
sort	samtools sort [-no] [-m maxMem] <in.bam> <out.prefix> Sort alignments by leftmost coordinates. File <out.prefix>.bam will be created. This command may also create temporary files <out.prefix>.%d.bam when the whole alignment cannot be fitted into memory (controlled by option -m). OPTIONS: -o Output the final alignment to the standard output. -n Sort by read names rather than by chromosomal coordinates -m INT Approximately the maximum required memory. [500000000]
merge	samtools merge [-nur1f] [-h inh.sam] [-R reg] <out.bam> <in1.bam> <in2.bam> [...] Merge multiple sorted alignments. The header reference lists of all the input BAM files, and the @SQ headers of inh.sam, if any, must all refer to the same set of reference sequences. The header reference list and (unless overridden by -h) ‘@’ headers of in1.bam will be copied to out.bam, and the headers of other files will be ignored. OPTIONS: -1 Use zlib compression level 1 to comrpess the output -f Force to overwrite the output file if present. -h FILE Use the lines of FILE as ‘@’ headers to be copied to out.bam, replacing any header lines that would otherwise be copied from in1.bam. (FILE is actually in SAM format, though any alignment records it may contain are ignored.) -n The input alignments are sorted by read names rather than by chromosomal coordinates -R STR Merge files in the specified region indicated by STR [null] -r Attach an RG tag to each alignment. The tag value is inferred from file names. -u Uncompressed BAM output
index	samtools index <aln.bam> Index sorted alignment for fast random access. Index file <aln.bam>.bai will be created.
idxstats	samtools idxstats <aln.bam> Retrieve and print stats in the index file. The output is TAB delimited with each line consisting of reference sequence name, sequence length, # mapped reads and # unmapped reads.
faidx	samtools faidx <ref.fasta> [region1 [...]] Index reference sequence in the FASTA format or extract subsequence from indexed reference sequence. If no region is specified, faidx will index the file and create<ref.fasta>.fai on the disk. If regions are speficified, the subsequences will be retrieved and printed to stdout in the FASTA format. The input file can be compressed in the RAZF format.
fixmate	samtools fixmate <in.nameSrt.bam> <out.bam> Fill in mate coordinates, ISIZE and mate related flags from a name-sorted alignment.
rmdup	samtools rmdup [-sS] <input.srt.bam> <out.bam> Remove potential PCR duplicates: if multiple read pairs have identical external coordinates, only retain the pair with highest mapping quality. In the paired-end mode, this command ONLY works with FR orientation and requires ISIZE is correctly set. It does not work for unpaired reads (e.g. two ends mapped to different chromosomes or orphan reads). OPTIONS: -s Remove duplicate for single-end reads. By default, the command works for paired-end reads only. -S Treat paired-end reads and single-end reads.
calmd	samtools calmd [-EeubSr] [-C capQcoef] <aln.bam> <ref.fasta> Generate the MD tag. If the MD tag is already present, this command will give a warning if the MD tag generated is different from the existing tag. Output SAM by default. OPTIONS: -A When used jointly with -r this option overwrites the original base quality. -e Convert a the read base to = if it is identical to the aligned reference base. Indel caller does not support the = bases at the moment. -u Output uncompressed BAM -b Output compressed BAM -S The input is SAM with header lines -C INT Coefficient to cap mapping quality of poorly mapped reads. See the pileupcommand for details. [0] -r Compute the BQ tag (without -A) or cap base quality by BAQ (with -A). -E Extended BAQ calculation. This option trades specificity for sensitivity, though the effect is minor.
targetcut	samtools targetcut [-Q minBaseQ] [-i inPenalty] [-0 em0] [-1 em1] [-2 em2] [-f ref] <in.bam> This command identifies target regions by examining the continuity of read depth, computes haploid consensus sequences of targets and outputs a SAM with each sequence corresponding to a target. When option -f is in use, BAQ will be applied. This command is only designed for cutting fosmid clones from fosmid pool sequencing [Ref. Kitzman et al. (2010)].

phase

samtools phase [-AF] [-k len] [-b prefix] [-q minLOD] [-Q minBaseQ] <in.bam> Call and phase heterozygous SNPs. OPTIONS: -A Drop reads with ambiguous phase. -b STR Prefix of BAM output. When this option is in use, phase-0 reads will be saved in fileSTR.0.bam and phase-1 reads in STR.1.bam. Phase unknown reads will be randomly allocated to one of the two files. Chimeric reads with switch errors will be saved inSTR.chimeric.bam. [null] -F Do not attempt to fix chimeric reads. -k INT Maximum length for local phasing. [13] -q INT Minimum Phred-scaled LOD to call a heterozygote. [40] -Q INT Minimum base quality to be used in het calling. [13]

BCFTOOLS COMMANDS AND OPTIONS

view	bcftools view [-AbFGNQSucgv] [-D seqDict] [-l listLoci] [-s listSample] [-igapSNPratio] [-t mutRate] [-p varThres] [-P prior] [-1 nGroup1] [-d minFrac] [-UnPerm] [-X permThres] [-T trioType] in.bcf [region] Convert between BCF and VCF, call variant candidates and estimate allele frequencies. Input/Output Options:  -A   Retain all possible alternate alleles at variant sites. By default, the view command discards unlikely alleles. -b Output in the BCF format. The default is VCF. -D FILE Sequence dictionary (list of chromosome names) for VCF->BCF conversion [null] -F Indicate PL is generated by r921 or before (ordering is different). -G Suppress all individual genotype information. -l FILE List of sites at which information are outputted [all sites] -N Skip sites where the REF field is not A/C/G/T -Q Output the QCALL likelihood format -s FILE List of samples to use. The first column in the input gives the sample names and the second gives the ploidy, which can only be 1 or 2. When the 2nd column is absent, the sample ploidy is assumed to be 2. In the output, the ordering of samples will be identical to the one in FILE. [null] -S The input is VCF instead of BCF. -u Uncompressed BCF output (force -b). Consensus/Variant Calling Options:  -c   Call variants using Bayesian inference. This option automatically invokes option -e. -d FLOAT When -v is in use, skip loci where the fraction of samples covered by reads is below FLOAT. [0] -e Perform max-likelihood inference only, including estimating the site allele frequency, testing Hardy-Weinberg equlibrium and testing associations with LRT. -g Call per-sample genotypes at variant sites (force -c) -i FLOAT Ratio of INDEL-to-SNP mutation rate [0.15] -p FLOAT A site is considered to be a variant if P(ref|D)<FLOAT [0.5] -P STR Prior or initial allele frequency spectrum. If STR can be full, cond2,flat or the file consisting of error output from a previous variant calling run. -t FLOAT Scaled muttion rate for variant calling [0.001] -T STR Enable pair/trio calling. For trio calling, option -s is usually needed to be applied to configure the trio members and their ordering. In the file supplied to the option -s, the first sample must be the child, the second the father and the third the mother. The valid values of STR are ‘pair’, ‘trioauto’, ‘trioxd’ and ‘trioxs’, where ‘pair’ calls differences between two input samples, and ‘trioxd’ (‘trioxs’) specifies that the input is from the X chromosome non-PAR regions and the child is a female (male). [null] -v Output variant sites only (force -c)：这里有-v这个选项就只输出snp位点，如果没有-v那么就是输出所有的位点（测序所包含的） Contrast Calling and Association Test Options:  -1 INT   Number of group-1 samples. This option is used for dividing the samples into two groups for contrast SNP calling or association test. When this option is in use, the following VCF INFO will be outputted: PC2, PCHI2 and QCHI2. [0] -U INT Number of permutations for association test (effective only with -1) [0] -X FLOAT Only perform permutations for P(chi^2)<FLOAT (effective only with -U) [0.01]
index	bcftools index in.bcf Index sorted BCF for random access.

cat	bcftools cat in1.bcf ["in2.bcf "[..."]]]" Concatenate BCF files. The input files are required to be sorted and have identical samples appearing in the same order.

SAM FORMAT

Sequence Alignment/Map (SAM) format is TAB-delimited. Apart from the header lines, which are started with the ‘@’ symbol, each alignment line consists of:

Col	Field	Description
1	QNAME	Query template/pair NAME
2	FLAG	bitwise FLAG
3	RNAME	Reference sequence NAME
4	POS	1-based leftmost POSition/coordinate of clipped sequence
5	MAPQ	MAPping Quality (Phred-scaled)
6	CIAGR	extended CIGAR string
7	MRNM	Mate Reference sequence NaMe (‘=’ if same as RNAME)
8	MPOS	1-based Mate POSistion
9	TLEN	inferred Template LENgth (insert size)
10	SEQ	query SEQuence on the same strand as the reference
11	QUAL	query QUALity (ASCII-33 gives the Phred base quality)
12+	OPT	variable OPTional fields in the format TAG:VTYPE:VALUE

Each bit in the FLAG field is defined as:

Flag	Chr	Description
0x0001	p	the read is paired in sequencing
0x0002	P	the read is mapped in a proper pair
0x0004	u	the query sequence itself is unmapped
0x0008	U	the mate is unmapped
0x0010	r	strand of the query (1 for reverse)
0x0020	R	strand of the mate
0x0040	1	the read is the first read in a pair
0x0080	2	the read is the second read in a pair
0x0100	s	the alignment is not primary
0x0200	f	the read fails platform/vendor quality checks
0x0400	d	the read is either a PCR or an optical duplicate

where the second column gives the string representation of the FLAG field.

VCF FORMAT

The Variant Call Format (VCF) is a TAB-delimited format with each data line consists of the following fields:

Col	Field	Description
1	CHROM	CHROMosome name
2	POS	the left-most POSition of the variant
3	ID	unique variant IDentifier
4	REF	the REFerence allele
5	ALT	the ALTernate allele(s), separated by comma
6	QUAL	variant/reference QUALity
7	FILTER	FILTers applied
8	INFO	INFOrmation related to the variant, separated by semi-colon
9	FORMAT	FORMAT of the genotype fields, separated by colon (optional)
10+	SAMPLE	SAMPLE genotypes and per-sample information (optional)

The following table gives the INFO tags used by samtools and bcftools.

Tag	Format	Description
AF1	double	Max-likelihood estimate of the site allele frequency (AF) of the first ALT allele
DP	int	Raw read depth (without quality filtering)
DP4	int[4]	# high-quality reference forward bases, ref reverse, alternate for and alt rev bases
FQ	int	Consensus quality. Positive: sample genotypes different; negative: otherwise
MQ	int	Root-Mean-Square mapping quality of covering reads
PC2	int[2]	Phred probability of AF in group1 samples being larger (,smaller) than in group2
PCHI2	double	Posterior weighted chi^2 P-value between group1 and group2 samples
PV4	double[4]	P-value for strand bias, baseQ bias, mapQ bias and tail distance bias
QCHI2	int	Phred-scaled PCHI2
RP	int	# permutations yielding a smaller PCHI2
CLR	int	Phred log ratio of genotype likelihoods with and without the trio/pair constraint
UGT	string	Most probable genotype configuration without the trio constraint
CGT	string	Most probable configuration with the trio constraint

EXAMPLES

o	Import SAM to BAM when @SQ lines are present in the header: samtools view -bS aln.sam > aln.bam If @SQ lines are absent: samtools faidx ref.fa  samtools view -bt ref.fa.fai aln.sam > aln.bam where ref.fa.fai is generated automatically by the faidx command.
o	Attach the RG tag while merging sorted alignments: perl -e ’print "@RG\tID:ga\tSM:hs\tLB:ga\tPL:Illumina\n@RG\tID:454\tSM:hs\tLB:454\tPL:454\n"’ > rg.txt  samtools merge -rh rg.txt merged.bam ga.bam 454.bam The value in a RG tag is determined by the file name the read is coming from. In this example, in the merged.bam, reads from ga.bam will be attached RG:Z:ga, while reads from454.bam will be attached RG:Z:454.
o	Call SNPs and short INDELs for one diploid individual: samtools mpileup -ugf ref.fa aln.bam | bcftools view -bvcg - > var.raw.bcf  bcftools view var.raw.bcf | vcfutils.pl varFilter -D 100 > var.flt.vcf The -D option of varFilter controls the maximum read depth, which should be adjusted to about twice the average read depth. One may consider to add -C50 to mpileup if mapping quality is overestimated for reads containing excessive mismatches. Applying this option usually helps BWA-short but may not other mappers.
o	Generate the consensus sequence for one diploid individual: samtools mpileup -uf ref.fa aln.bam | bcftools view -cg - | vcfutils.pl vcf2fq > cns.fq
o	Call somatic mutations from a pair of samples: samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair - > var.bcf In the output INFO field, CLR gives the Phred-log ratio between the likelihood by treating the two samples independently, and the likelihood by requiring the genotype to be identical. This CLR is effectively a score measuring the confidence of somatic calls. The higher the better.
o	Call de novo and somatic mutations from a family trio: samtools mpileup -DSuf ref.fa aln.bam | bcftools view -bvcgT pair -s samples.txt - > var.bcf File samples.txt should consist of three lines specifying the member and order of samples (in the order of child-father-mother). Similarly, CLR gives the Phred-log likelihood ratio with and without the trio constraint. UGT shows the most likely genotype configuration without the trio constraint, and CGT gives the most likely genotype configuration satisfying the trio constraint.
o	Phase one individual: samtools calmd -AEur aln.bam ref.fa | samtools phase -b prefix - > phase.out The calmd command is used to reduce false heterozygotes around INDELs.
o	Call SNPs and short indels for multiple diploid individuals: samtools mpileup -P ILLUMINA -ugf ref.fa *.bam | bcftools view -bcvg - > var.raw.bcf  bcftools view var.raw.bcf | vcfutils.pl varFilter -D 2000 > var.flt.vcf Individuals are identified from the SM tags in the @RG header lines. Individuals can be pooled in one alignment file; one individual can also be separated into multiple files. The-P option specifies that indel candidates should be collected only from read groups with the @RG-PL tag set to ILLUMINA. Collecting indel candidates from reads sequenced by an indel-prone technology may affect the performance of indel calling.
o	Derive the allele frequency spectrum (AFS) on a list of sites from multiple individuals: samtools mpileup -Igf ref.fa *.bam > all.bcf  bcftools view -bl sites.list all.bcf > sites.bcf  bcftools view -cGP cond2 sites.bcf > /dev/null 2> sites.1.afs  bcftools view -cGP sites.1.afs sites.bcf > /dev/null 2> sites.2.afs  bcftools view -cGP sites.2.afs sites.bcf > /dev/null 2> sites.3.afs  ...... where sites.list contains the list of sites with each line consisting of the reference sequence name and position. The following bcftools commands estimate AFS by EM.
o	Dump BAQ applied alignment for other SNP callers: samtools calmd -bAr aln.bam > aln.baq.bam It adds and corrects the NM and MD tags at the same time. The calmd command also comes with the -C option, the same as the one in pileup and mpileup. Apply if it helps.

LIMITATIONS

o	Unaligned words used in bam_import.c, bam_endian.h, bam.c and bam_aux.c.
o	Samtools paired-end rmdup does not work for unpaired reads (e.g. orphan reads or ends mapped to different chromosomes). If this is a concern, please use Picard’s MarkDuplicate which correctly handles these cases, although a little slower.

AUTHOR

Heng Li from the Sanger Institute wrote the C version of samtools. Bob Handsaker from the Broad Institute implemented the BGZF library and Jue Ruan from Beijing Genomics Institute wrote the RAZF library. John Marshall and Petr Danecek contribute to the source code and various people from the 1000 Genomes Project have contributed to the SAM format specification.

SEE ALSO

Samtools website: <http://samtools.sourceforge.net>

samtools-0.1.17

samtools (1)

05 July 2011

上面的内容参考了一下网址的内容

http://www.plob.org/2012/02/04/1703.html

http://www.plob.org/2012/02/04/1700.html

http://www.hudsonalpha.org/gsl/software/bam2fastq.php

http://liucheng.name/670/

http://samtools.sourceforge.net/samtools.shtml

http://blog.sina.com.cn/s/blog_4af3f0d20100xvq1.html