在转录组数据与参考基因组进行比对后，得到sam文件，后续分析需要将sam转换为bam，这里用到的工具是SAMtools。
序列比对 —— Hisat2 - 简书 (jianshu.com)
SAMtools的主要功能是读取、写出、编辑、查看SAM/BAM/CRAM格式的数据文件。
SAMtools官方网站：
http://www.htslib.org/
1.下载安装
链接：
http://www.htslib.org/download/
$ tar -jxvf samtools-1.14.tar.bz2
安装：
$ cd ~/samtools-1.14
$ ./configure --prefix=/your/path
$ make
$ make install
2. 基础命令
$ samtools
Program: samtools (Tools for alignments in the SAM format)
Version: 1.14 (using htslib 1.14)
Usage: samtools <command> [options]
Commands:
-- Indexing
dict create a sequence dictionary file
faidx index/extract FASTA
fqidx index/extract FASTQ
index index alignment
-- Editing
calmd recalculate MD/NM tags and '=' bases
fixmate fix mate information
reheader replace BAM header
targetcut cut fosmid regions (for fosmid pool only)
addreplacerg adds or replaces RG tags
markdup mark duplicates
ampliconclip clip oligos from the end of reads
-- 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
import Converts FASTA or FASTQ files to SAM/BAM/CRAM
-- Statistics
bedcov read depth per BED region
coverage alignment depth and percent coverage
depth compute the depth
flagstat simple stats
idxstats BAM index stats
phase phase heterozygotes
stats generate stats (former bamcheck)
ampliconstats generate amplicon specific stats
-- Viewing
flags explain BAM flags
tview text alignment viewer
view SAM<->BAM<->CRAM conversion
depad convert padded BAM to unpadded BAM
samples list the samples in a set of SAM/BAM/CRAM files
-- Misc
help [cmd] display this help message or help for [cmd]
version detailed version information
这里看到基础命令主要分为六个模块，分别为索引、编辑、文件操作、统计、视图以及其他。这里想要SAM格式转为BAM格式，主要用到的是Viewing模块中的view。
3. sam格式转换为bam
说明书：
http://www.htslib.org/workflow/fastq.html
在老版本1.9的samtools中，需要用-s 指定sam文件，1.14中不需要指定。
samtools view命令完成sam转为bam。
3.1 view命令基础功能
$ samtools view
Usage: samtools view [options] <in.bam>|<in.sam>|<in.cram> [region ...]
Output options:
-b, --bam Output BAM
-C, --cram Output CRAM (requires -T)
-1, --fast Use fast BAM compression (implies --bam)
-u, --uncompressed Uncompressed BAM output (implies --bam)
-h, --with-header Include header in SAM output
-H, --header-only Print SAM header only (no alignments)
--no-header Print SAM alignment records only [default]
-c, --count Print only the count of matching records
-o, --output FILE Write output to FILE [standard output]
-U, --unoutput FILE, --output-unselected FILE
Output reads not selected by filters to FILE
-p, --unmap Set flag to UNMAP on reads not selected
then write to output file.
Input options:
-t, --fai-reference FILE FILE listing reference names and lengths
-M, --use-index Use index and multi-region iterator for regions
--region[s]-file FILE Use index to include only reads overlapping FILE
-X, --customized-index Expect extra index file argument after <in.bam>
Filtering options (Only include in output reads that...):
-L, --target[s]-file FILE ...overlap (BED) regions in FILE
-r, --read-group STR ...are in read group STR
-R, --read-group-file FILE ...are in a read group listed in FILE
-N, --qname-file FILE ...whose read name is listed in FILE
-d, --tag STR1[:STR2] ...have a tag STR1 (with associated value STR2)
-D, --tag-file STR:FILE ...have a tag STR whose value is listed in FILE
-q, --min-MQ INT ...have mapping quality >= INT
-l, --library STR ...are in library STR
-m, --min-qlen INT ...cover >= INT query bases (as measured via CIGAR)
-e, --expr STR ...match the filter expression STR
-f, --require-flags FLAG ...have all of the FLAGs present
-F, --excl[ude]-flags FLAG ...have none of the FLAGs present
-G FLAG EXCLUDE reads with all of the FLAGs present
--subsample FLOAT Keep only FLOAT fraction of templates/read pairs
--subsample-seed INT Influence WHICH reads are kept in subsampling [0]
-s INT.FRAC Same as --subsample 0.FRAC --subsample-seed INT
Processing options:
--add-flags FLAG Add FLAGs to reads
--remove-flags FLAG Remove FLAGs from reads
-x, --remove-tag STR
Comma-separated read tags to strip (repeatable) [null]
--keep-tag STR
Comma-separated read tags to preserve (repeatable) [null].
Equivalent to "-x ^STR"
-B, --remove-B Collapse the backward CIGAR operation
General options:
-?, --help Print long help, including note about region specification
-S Ignored (input format is auto-detected)
--no-PG Do not add a PG line
--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]
-@, --threads INT
Number of additional threads to use [0]
--write-index
Automatically index the output files [off]
--verbosity INT
Set level of verbosity
3.2 格式转换
$ samtools view -@8 -b LPF1_R1_MP.sam > LPF1_R1_MP.bam
-@8：8个线程
-b：输出格式bam文件 >：输出文件名
如果是1.9版本的SAMtools可以参考这篇文章：
https://www.jianshu.com/p/6b7a442d293f
引用转载请注明出处，如有错误敬请指出。
