Hisat2 bowtie2比对结果解读（Hisat2 Alignment summary）

RNA-seq数据的比对结果怎么解读？网上有很多人问，这里做一个大致的总结。

Hisat2和bowtie2比对后产生的Alignment summary的格式是一样的，如下：

Alignment summary

When HISAT2 finishes running, it prints messages summarizing what happened. These messages are printed to the "standard error" ("stderr") filehandle. For datasets consisting of unpaired reads, the summary might look like this:

单端数据比对的结果如下：

20000 reads; of these:
  20000 (100.00%) were unpaired; of these:
    1247 (6.24%) aligned 0 times
    18739 (93.69%) aligned exactly 1 time
    14 (0.07%) aligned >1 times
93.77% overall alignment rate

For datasets consisting of pairs, the summary might look like this:

双端数据比对的结果如下：

10000 reads; of these:
  10000 (100.00%) were paired; of these:
    650 (6.50%) aligned concordantly 0 times
    8823 (88.23%) aligned concordantly exactly 1 time
    527 (5.27%) aligned concordantly >1 times
    ----
    650 pairs aligned concordantly 0 times; of these:
      34 (5.23%) aligned discordantly 1 time
    ----
    616 pairs aligned 0 times concordantly or discordantly; of these:
      1232 mates make up the pairs; of these:
        660 (53.57%) aligned 0 times
        571 (46.35%) aligned exactly 1 time
        1 (0.08%) aligned >1 times
96.70% overall alignment rate

（copy自：Hisat2官网）

单端的就没什么好说的了，主要看双端序列比对：

650 (6.50%) aligned concordantly 0 times 是什么意思？ 其实第一部分描述的是pair-end模式下的一致比对结果，aligned concordantly就是read1和read2同时合理的比对到了基因组/转录组上。

8823 (88.23%) aligned concordantly exactly 1 time，exactly 1 time 就是只有一种比对结果。>1 times 就是read1和read2可以同时比对到多个地方。

第二部分，pair-end模式下不一致的比对结果。

650 pairs aligned concordantly 0 times; of these: 34 (5.23%) aligned discordantly 1 time

concordantly 0 times就是read1和read2不能同时合理的同时比对到基因组/转录组上，aligned discordantly 1 time 最难理解。

以下是bowtie2官网对 aligned discordantly 的解释：

Concordant pairs match pair expectations, discordant pairs don’t

A pair that aligns with the expected relative mate orientation and with the expected range of distances between mates is said to align “concordantly”. If both mates have unique alignments, but the alignments do not match paired-end expectations (i.e. the mates aren’t in the expected relative orientation, or aren’t within the expected distance range, or both), the pair is said to align “discordantly”. Discordant alignments may be of particular interest, for instance, when seeking structural variants.

The expected relative orientation of the mates is set using the --ff, --fr, or --rf options. The expected range of inter-mates distances (as measured from the furthest extremes of the mates; also called “outer distance”) is set with the -I and -X options. Note that setting -I and -X far apart makes Bowtie 2 slower. See documentation for -I and -X.

To declare that a pair aligns discordantly, Bowtie 2 requires that both mates align uniquely. This is a conservative threshold, but this is often desirable when seeking structural variants.

By default, Bowtie 2 searches for both concordant and discordant alignments, though searching for discordant alignments can be disabled with the --no-discordantoption.

Mixed mode: paired where possible, unpaired otherwise

If Bowtie 2 cannot find a paired-end alignment for a pair, by default it will go on to look for unpaired alignments for the constituent mates. This is called “mixed mode.” To disable mixed mode, set the --no-mixed option.

Bowtie 2 runs a little faster in --no-mixed mode, but will only consider alignment status of pairs per se, not individual mates.

重点：If both mates have unique alignments, but the alignments do not match paired-end expectations (i.e. the mates aren’t in the expected relative orientation, or aren’t within the expected distance range, or both), the pair is said to align “discordantly”.

discordantly比对就是read1和read2都能比对上，但是不合理，

1. 比对方向不对，pair-end测序的方向是固定的；

2.read1和read2的插入片段长度是有限的

第三部分就是对剩余reads（既不能concordantly，也不能discordantly 1 time）的单端模式的比对，没什么好说的。

以上的理解来自SEQanswers：

16182999 reads; of these:
16182999 (100.00%) were paired; of these:
5731231 (35.42%) aligned concordantly 0 times
4522376 (27.95%) aligned concordantly exactly 1 time
5929392 (36.64%) aligned concordantly >1 times
----
5731231 pairs aligned concordantly 0 times; of these:
2381431 (41.55%) aligned discordantly 1 time
----
3349800 pairs aligned 0 times concordantly or discordantly; of these:
6699600 mates make up the pairs; of these:
3814736 (56.94%) aligned 0 times
1883429 (28.11%) aligned exactly 1 time
1001435 (14.95%) aligned >1 times
88.21% overall alignment rate

the Bowtie2 result summary is divided in 3 sections:
Concordant alignment - In your data (4522376 + 5929392) reads align concordantly. Which is 64.59% of reads
Discordant alignment - So now 5731231 reads remain which is 35.41% (100-64.59). Of these, 2381431 reads align discordantly. That is to say, of the non-concordant fraction, 41.55% of reads (2381431 reads) align discordantly.
The rest - Now, remember that alignment whether concord. or discord., but both are aligned in paired-end mode. The rest of the reads either align as singles (i.e. Read1 in one locus & Read2 in completely different locus or one mate aligned and the other unaligned) or may not align at all. So the reads that are in this section is Total -(Concord.+Discord.). That is 16182999 -(10451768+2381431) = 3349800 reads.
Since alignment, if any, here is in single fashion so we calculate in mates (readsx2).

Now to reach the overall alignment, count the mates in total (i.e. mates aligned in paired and mates aligned in single fashion). That would be -
(10451768 x2)+(2381431 x2)+1883429+1001435 = 28551262 mates
That is 28551262 mates aligned of total (16182999 x2) mates, which is 88.21%.

为什么要区分这么多比对类型，简单点不好吗？no

在具体项目分析时你就会体会到这些比对类型信息的重要性，有时间再讲。see you next time.