Difference between Hard Clip（H） and Soft Clip（S） in Samtools CIGAR string

一般人都知道 H 和 S 的表面上的区别，即 S 就是 soft， H 就是 hard，S 后，序列里还是会保留序列的信息，而 H 则不会。

-------------------------------------------后面都不用看了，H和S没有区别，比对软件不能发现嵌合体--------------------------------------

但这只是表面上的，在深层次的意义上， H 和 S 又有什么本质的不同呢？

首先要了解嵌合体的概念：

嵌合体就是两个不同的序列错误的拼接到了一起，也就是一条序列分别比对到了 ref 的两个地方（这和多重比对、次级比对之间又有区别）

Example of extended CIGAR and the pileup output.

(a) Alignments of one pair of reads and three single-end reads.

(b) The corresponding SAM file. The ‘@SQ’ line in the header section gives the order of reference sequences. Notably, r001 is the name of a read pair. According to FLAG 163 (=1 + 2 + 32 + 128), the read mapped to position 7 is the second read in the pair (128) and regarded as properly paired (1 + 2); its mate is mapped to 37 on the reverse strand (32). Read r002 has three soft-clipped (unaligned) bases. The coordinate shown in SAM is the position of the first aligned base. The CIGAR string for this alignment contains a P (padding) operation which correctly aligns the inserted sequences. Padding operations can be absent when an aligner does not support multiple sequence alignment. The last six bases of read r003 map to position 9, and the first five to position 29 on the reverse strand. The hard clipping operation H indicates that the clipped sequence is not present in the sequence field. The NM tag gives the number of mismatches. Read r004 is aligned across an intron, indicated by the N operation.

(c) Simplified pileup output by SAMtools. Each line consists of reference name, sorted coordinate, reference base, the number of reads covering the position and read bases. In the fifth field, a dot or a comma denotes a base identical to the reference; a dot or a capital letter denotes a base from a read mapped on the forward strand, while a comma or a lowercase letter on the reverse strand.

clipped alignment因为着在比对过程中，并没有用到全部的read的序列，read两段的序列被截取了（clip or trim）。如下表示，即为clip alignment。

Alignment:
Read:          ACGGTTGCGTTAA-TCCGCCACG
|                           ||||||||| ||||||
Reference: TAACTTGCGTTAAATCCGCCTGG

与clipped alignment对应的是spliced alignment，即read的中间没有比对到而两段比对上了。对应的表示如下：

Alignment:
Read:          ACGGTTGCGTTAAGCTCATCCGCCACG
|                 |||||||||||||         |||||||||
Reference: ACGGTTGCGTTAA…..TCCGCCACG

clip alignment对应的CIGAR表示有两种S （soft clip） 和H （hard clip）。
BWA提到If the read has a chimeric alignment, the paired or the top hit uses soft clipping and is marked with neither 0x800 nor 0x100 bits. All the other hits part of the chimeric alignment will use hard clipping and be marked with 0x800 if option “-M” is not in use, or marked with 0x100 otherwise.

即如果发现嵌合比对，最好的比对top hit标记为soft clipping，其余的则标记为hard clipping。

如果是hard clip，则截取的部分不会在SAM文件对应的read中出现 (clipped sequences not present in SEQ)，如果是soft clip (clipped sequences present in SEQ)，则会出现。

Understand？

Ref：https://github.com/lh3/bwa/blob/master/NEWS.md

理解1：

Hard masked bases do not appear in the SEQ string, soft masked bases do.

So, if your cigar is: `10H10M10H` then the SEQ will only be 10 bases long.

if your cigar is 10S10M10S then the SEQ and base-quals will be 30 bases long.

首先，结果展示方式有区别：​比如说10H10M10H，第10列的碱基序列只显示10bp；而如果是10S10M10S的话，就会显示30bp的序列，尽管开头和结尾的20bp也没比上。

In the case of soft-masking, even though the SEQ is present, it is not used by variant callers and not displayed when you view your data in a viewer. In either case, masked bases should not be used in calculating coverage.

在soft中，即使显示的序列比hard的要长，但是计算变异或可视化比对结果时，这些序列也不会被考虑。而且，2种情况计算覆盖度​时，mask的碱基都不会考虑。

例子：

20692128    97    viral_genome    21417    60    69M32S    chr7    101141242    0    TACATCTTCTCCCTCTCTCACGACACAAGAATTAGTCACATAGGGATGTTCTCGTAAATCTACATTATCTTACAAAAACATTTTTTAAAAATTTGCTAGGT （101bp）    GGGGGGGGGGGGGGEGGEGGGGGGGGGFGGGGGGGGGGGGGEGFFGGGGGGGFGGFGGGGEGGGGGGGGGGGEGEFFGGGFEGGGGGFGCGGGFBGGGBG@    NM:i:4    MD:Z:6G34G6C5C14    AS:i:49    XS:i:0    SA:Z:chr7,101141091,+,66S35M,60,0;

20692128    353    chr7    101141091    60    66H35M    =    101141242    252    ATCTTACAAAAACATTTTTTAAAAATTTGCTAGGT  （35bp）GGGGGGEGEFFGGGFEGGGGGFGCGGGFBGGGBG@    NM:i:0    MD:Z:35    AS:i:35    XS:i:23    SA:Z:gi|224020395|ref|NC_001664.2|,21417,+,69M32S,60,4;

20692128    145    chr7    101141242    60    101M    gi|224020395|ref|NC_001664.2|    21417    0    GCAACAGAGCGAGACCCTATATTCATGAGTGTTGCAATGAGCCAAGTAGTGGAGGTTGGCTTTTGAAGGCAGAAAAGGACTGAGAAAAGCTAACACAGAGA    FEGCGGGGGCGEFCDEEEEGGGGGGGGGGGGGGGEGGGGGGFGGGEGGG

​理解2：

当同一条reads比对到不同chr时（嵌合reads），会以hard clip的显示显示。比如上面的例子，R1分别比到了viral基因组和chr7上（前面69bp比到viral，后面35比到chr7），R2比到了chr7。

sam格式详细说明见：http://davetang.org/wiki/tiki-index.php?page=SAM