Hisat2与STAR一样，是一款比对软件。它使用了基于BWT和Ferragina-manzini (Fm) index 两种算法的索引框架。Hiasat2采用了新的比对策略：

1. RNA-seq产生的reads可能跨长度比较大的内含子，哺乳动物中甚至最长能达到1MB，同时外显子比较短，read也比较短，会有很多read（模拟数据中大概34%）跨两个外显子的情况
2. 为了更好的比对，将跨外显子的reads分成了三类：1）长锚定read，至少有16bp在两个外显子的每一个上 2）中间锚定read，有8-15bp在一个外显子上 3）短锚定read，只有1-7bp在一个外显子上
3. 所以总的reads可以被划分为五类：1）不跨外显子的read 2）长锚定read 3）中间锚定read 4）短锚定read 5）跨两个外显子以上的read
4. 在模拟的数据中，有25%左右的read是长锚定read，这种read在大多数情况下可以被唯一的定位到人的基因组上
5. 5%为中间锚定read，对于这类，很多依赖于全局索引的算法就很难执行下去（需要比对很多次），而hisat，可以先将read中的长片段实现唯一比对，之后再使用局部索引对剩下的小片段进行比对（局部索引可以实现快速检索）
6. 4.2%为短锚定read，因为这些序列特别短，因此只能通过在hisat比对其它read时发现的剪切位点或者用户自己提供的剪切位点来辅助比对
7. 最后还有3%的是跨多个外显子的read，比对策略在hisat的online method中有介绍，文章中没有详解
8. 比对过程中，中间锚定read、短锚定read、跨多个外显子read的比对占总比对时长的30%-60%，而且比对错误率很高！
   下面介绍一下Hisat2的使用。

安装

依然是使用conda进行安装，方便省事。conda install hisat2

建立Hisat2索引

使用Hisat2进行比对需要应用Hisat2软件的索引，建立索引需要依据基因组文件，使用一下命令可建立索引。

##后台运行
nohup hisat2-build -p 10 Homo_sapiens.GRCh38.dna.primary_assembly.fa /mnt/f/index/hisat2_index/homo &

##看一下hisat2-build的参数设置
hisat2-build -h
HISAT2 version 2.2.1 by Daehwan Kim (infphilo@gmail.com, http://www.ccb.jhu.edu/people/infphilo)
Usage: hisat2-build [options]* <reference_in> <ht2_index_base>
    reference_in            comma-separated list of files with ref sequences
    hisat2_index_base       write ht2 data to files with this dir/basename
Options:
    -c                      reference sequences given on cmd line (as
                            <reference_in>)
    --large-index           force generated index to be 'large', even if ref
                            has fewer than 4 billion nucleotides
    -a/--noauto             disable automatic -p/--bmax/--dcv memory-fitting
    -p <int>                number of threads
    --bmax <int>            max bucket sz for blockwise suffix-array builder
    --bmaxdivn <int>        max bucket sz as divisor of ref len (default: 4)
    --dcv <int>             diff-cover period for blockwise (default: 1024)
    --nodc                  disable diff-cover (algorithm becomes quadratic)
    -r/--noref              don't build .3/.4.ht2 (packed reference) portion
    -3/--justref            just build .3/.4.ht2 (packed reference) portion
    -o/--offrate <int>      SA is sampled every 2^offRate BWT chars (default: 5)
    -t/--ftabchars <int>    # of chars consumed in initial lookup (default: 10)
    --localoffrate <int>    SA (local) is sampled every 2^offRate BWT chars (default: 3)
    --localftabchars <int>  # of chars consumed in initial lookup in a local index (default: 6)
    --snp <path>            SNP file name
    --haplotype <path>      haplotype file name
    --ss <path>             Splice site file name
    --exon <path>           Exon file name
    --repeat-ref <path>     Repeat reference file name
    --repeat-info <path>    Repeat information file name
    --repeat-snp <path>     Repeat snp file name
    --repeat-haplotype <path>   Repeat haplotype file name
    --seed <int>            seed for random number generator
    -q/--quiet              disable verbose output (for debugging)
    -h/--help               print detailed description of tool and its options
    --usage                 print this usage message
    --version               print version information and quit

可得到以下文件

比对

建立好索引后，即基于此对质控好的clean data进行比对啦！
使用以下代码进行批量比对。

##看一下用法
hisat2 -h
HISAT2 version 2.2.1 by Daehwan Kim (infphilo@gmail.com, www.ccb.jhu.edu/people/infphilo)
Usage:
  hisat2 [options]* -x <ht2-idx> {-1 <m1> -2 <m2> | -U <r>} [-S <sam>]

  <ht2-idx>  Index filename prefix (minus trailing .X.ht2).
  <m1>       Files with #1 mates, paired with files in <m2>.
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <m2>       Files with #2 mates, paired with files in <m1>.
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <r>        Files with unpaired reads.
             Could be gzip'ed (extension: .gz) or bzip2'ed (extension: .bz2).
  <sam>      File for SAM output (default: stdout)

  <m1>, <m2>, <r> can be comma-separated lists (no whitespace) and can be
  specified many times.  E.g. '-U file1.fq,file2.fq -U file3.fq'.

Options (defaults in parentheses):

 Input:
  -q                 query input files are FASTQ .fq/.fastq (default)
  --qseq             query input files are in Illumina's qseq format
  -f                 query input files are (multi-)FASTA .fa/.mfa
  -r                 query input files are raw one-sequence-per-line
  -c                 <m1>, <m2>, <r> are sequences themselves, not files
  -s/--skip <int>    skip the first <int> reads/pairs in the input (none)
  -u/--upto <int>    stop after first <int> reads/pairs (no limit)
  -5/--trim5 <int>   trim <int> bases from 5'/left end of reads (0)
  -3/--trim3 <int>   trim <int> bases from 3'/right end of reads (0)
  --phred33          qualities are Phred+33 (default)
  --phred64          qualities are Phred+64
  --int-quals        qualities encoded as space-delimited integers

 Presets:                 Same as:
   --fast                 --no-repeat-index
   --sensitive            --bowtie2-dp 1 -k 30 --score-min L,0,-0.5
   --very-sensitive       --bowtie2-dp 2 -k 50 --score-min L,0,-1

 Alignment:
  --bowtie2-dp <int> use Bowtie2's dynamic programming alignment algorithm (0) - 0: no dynamic programming, 1: conditional dynamic programming, and 2: unconditional dynamic programming (slowest)
  --n-ceil <func>    func for max # non-A/C/G/Ts permitted in aln (L,0,0.15)
  --ignore-quals     treat all quality values as 30 on Phred scale (off)
  --nofw             do not align forward (original) version of read (off)
  --norc             do not align reverse-complement version of read (off)
  --no-repeat-index  do not use repeat index

 Spliced Alignment:
  --pen-cansplice <int>              penalty for a canonical splice site (0)
  --pen-noncansplice <int>           penalty for a non-canonical splice site (12)
  --pen-canintronlen <func>          penalty for long introns (G,-8,1) with canonical splice sites
  --pen-noncanintronlen <func>       penalty for long introns (G,-8,1) with noncanonical splice sites
  --min-intronlen <int>              minimum intron length (20)
  --max-intronlen <int>              maximum intron length (500000)
  --known-splicesite-infile <path>   provide a list of known splice sites
  --novel-splicesite-outfile <path>  report a list of splice sites
  --novel-splicesite-infile <path>   provide a list of novel splice sites
  --no-temp-splicesite               disable the use of splice sites found
  --no-spliced-alignment             disable spliced alignment
  --rna-strandness <string>          specify strand-specific information (unstranded)
  --tmo                              reports only those alignments within known transcriptome
  --dta                              reports alignments tailored for transcript assemblers
  --dta-cufflinks                    reports alignments tailored specifically for cufflinks
  --avoid-pseudogene                 tries to avoid aligning reads to pseudogenes (experimental option)
  --no-templatelen-adjustment        disables template length adjustment for RNA-seq reads

 Scoring:
  --mp <int>,<int>   max and min penalties for mismatch; lower qual = lower penalty <6,2>
  --sp <int>,<int>   max and min penalties for soft-clipping; lower qual = lower penalty <2,1>
  --no-softclip      no soft-clipping
  --np <int>         penalty for non-A/C/G/Ts in read/ref (1)
  --rdg <int>,<int>  read gap open, extend penalties (5,3)
  --rfg <int>,<int>  reference gap open, extend penalties (5,3)
  --score-min <func> min acceptable alignment score w/r/t read length
                     (L,0.0,-0.2)

 Reporting:
  -k <int>           It searches for at most <int> distinct, primary alignments for each read. Primary alignments mean
                     alignments whose alignment score is equal to or higher than any other alignments. The search terminates
                     when it cannot find more distinct valid alignments, or when it finds <int>, whichever happens first.
                     The alignment score for a paired-end alignment equals the sum of the alignment scores of
                     the individual mates. Each reported read or pair alignment beyond the first has the SAM ‘secondary’ bit
                     (which equals 256) set in its FLAGS field. For reads that have more than <int> distinct,
                     valid alignments, hisat2 does not guarantee that the <int> alignments reported are the best possible
                     in terms of alignment score. Default: 5 (linear index) or 10 (graph index).
                     Note: HISAT2 is not designed with large values for -k in mind, and when aligning reads to long,
                     repetitive genomes, large -k could make alignment much slower.
  --max-seeds <int>  HISAT2, like other aligners, uses seed-and-extend approaches. HISAT2 tries to extend seeds to
                     full-length alignments. In HISAT2, --max-seeds is used to control the maximum number of seeds that
                     will be extended. For DNA-read alignment (--no-spliced-alignment), HISAT2 extends up to these many seeds
                     and skips the rest of the seeds. For RNA-read alignment, HISAT2 skips extending seeds and reports
                     no alignments if the number of seeds is larger than the number specified with the option,
                     to be compatible with previous versions of HISAT2. Large values for --max-seeds may improve alignment
                     sensitivity, but HISAT2 is not designed with large values for --max-seeds in mind, and when aligning
                     reads to long, repetitive genomes, large --max-seeds could make alignment much slower.
                     The default value is the maximum of 5 and the value that comes with -k times 2.
  -a/--all           HISAT2 reports all alignments it can find. Using the option is equivalent to using both --max-seeds
                     and -k with the maximum value that a 64-bit signed integer can represent (9,223,372,036,854,775,807).
  --repeat           report alignments to repeat sequences directly

 Paired-end:
  -I/--minins <int>  minimum fragment length (0), only valid with --no-spliced-alignment
  -X/--maxins <int>  maximum fragment length (500), only valid with --no-spliced-alignment
  --fr/--rf/--ff     -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)
  --no-mixed         suppress unpaired alignments for paired reads
  --no-discordant    suppress discordant alignments for paired reads

 Output:
  -t/--time          print wall-clock time taken by search phases
  --un <path>           write unpaired reads that didn't align to <path>
  --al <path>           write unpaired reads that aligned at least once to <path>
  --un-conc <path>      write pairs that didn't align concordantly to <path>
  --al-conc <path>      write pairs that aligned concordantly at least once to <path>
  (Note: for --un, --al, --un-conc, or --al-conc, add '-gz' to the option name, e.g.
  --un-gz <path>, to gzip compress output, or add '-bz2' to bzip2 compress output.)
  --summary-file <path> print alignment summary to this file.
  --new-summary         print alignment summary in a new style, which is more machine-friendly.
  --quiet               print nothing to stderr except serious errors
  --met-file <path>     send metrics to file at <path> (off)
  --met-stderr          send metrics to stderr (off)
  --met <int>           report internal counters & metrics every <int> secs (1)
  --no-head             suppress header lines, i.e. lines starting with @
  --no-sq               suppress @SQ header lines
  --rg-id <text>        set read group id, reflected in @RG line and RG:Z: opt field
  --rg <text>           add <text> ("lab:value") to @RG line of SAM header.
                        Note: @RG line only printed when --rg-id is set.
  --omit-sec-seq        put '*' in SEQ and QUAL fields for secondary alignments.

 Performance:
  -o/--offrate <int> override offrate of index; must be >= index's offrate
  -p/--threads <int> number of alignment threads to launch (1)
  --reorder          force SAM output order to match order of input reads
  --mm               use memory-mapped I/O for index; many 'hisat2's can share

 Other:
  --qc-filter        filter out reads that are bad according to QSEQ filter
  --seed <int>       seed for random number generator (0)
  --non-deterministic seed rand. gen. arbitrarily instead of using read attributes
  --remove-chrname   remove 'chr' from reference names in alignment
  --add-chrname      add 'chr' to reference names in alignment
  --version          print version information and quit
  -h/--help          print this usage message

##用一下代码批量分析
写一个bash脚本
vim mapping.sh
##把下面的代码复制进去
cat SRR_Acc_List.txt | while read sample
do
    hisat2 -p 10 -x /mnt/f/index/hisat2_index/homo/homo -1 /mnt/f/RNA-Seq/GSE176393（SRP323246）/1-cleandata/${sample}_clean_1.fastq.gz -2 /mnt/f/RNA-Seq/GSE176393（SRP323246）/1-cleandata/${sample}_clean_2.fastq.gz -S /mnt/f/RNA-Seq/GSE176393（SRP323246）/2-mapping/${sample}.sam
done

##后台运行脚本
nohup bash mapping.sh &

得到以下结果

比对后的结果.sam文件太占空间，我们将其转换为.bam文件，我们使用samtool进行。（后续文章将对samtool详细介绍）

#依然是批量后台运行
nohup cat SRR_Acc_List.txt | while read line; do samtools view -Sb 2-mapping/$line.sam > 2-mapping/$line.bam; done &

得到以下结果就可以暂时告一段落了。

这就是比对了，接下来我们就要详细学习一下HTSeq定量了