劉小澤寫于2020.4.6
記錄一個(gè)小知識(shí)點(diǎn)突琳，比對(duì)后SAM/BAM的CIGAR這一列中的含義北苟，重點(diǎn)是soft clipping 和 hard clipping的理解
首先了解SAM/BAM中的CIGAR含義
看這篇：再次理解SAM/BAM操作
根據(jù)sam的幫助文檔：http://samtools.sourceforge.net/SAM1.pdf
op Description
M Alignment match (can be a sequence match or mismatch
I Insertion to the reference
D Deletion from the reference
N Skipped region from the reference
S Soft clip on the read (clipped sequence present in <seq>)
H Hard clip on the read (clipped sequence NOT present in <seq>)
P Padding (silent deletion from the padded reference sequence)
然后來看什么是clipped alignment和spliced alignment
-
clipped alignment：read只有中間部分能比對(duì)上型型，而兩側(cè)在比對(duì)過程中被忽略
# 3S4M1D5M3S(3 soft, 4 match, 1 deletion, 5 match and 3 soft) Read: CGATTGC-TCCGCCAGG | |||| |||||| Ref:CCGATCGAGACTTGCGTCCGCCTCCCGATCA -
spliced alignment：read一端比對(duì)上硕勿，跳過了中間，然后另一端也比對(duì)上【CIGAR中用“N”表示】如果是轉(zhuǎn)錄組數(shù)據(jù)的比對(duì)結(jié)果，N表示內(nèi)含子极阅；其他類型組學(xué)數(shù)據(jù)中N也可以用涨享，但沒有意義筋搏，和deletion一個(gè)意思（參考：https://www.biostars.org/p/96347/）
# 3M1D4M13N5M # This only makes sense when you're aligning things like cDNA/expression data. REF: ATCGATCGATCGATCGATCGATCGATCGATCG |||||||||||||||||||||||||| QUERY: ATC-ATCG-------------ATCAT # 如果是DNA測(cè)序reads的比對(duì)奔脐，也可以全部用D來表示：即3M1D4M13D3M
然后clipped alignment有兩種形式
分為soft和hard（在SAM/BAM的CIGAR列分別用“S”和“H”表示），它們很相似吁讨，不同是：
soft-clipped: bases in 5' and 3' of the read are NOT part of the alignment. 這部分沒比對(duì)上但保留在了SAM/BAM比對(duì)結(jié)果中
-
hard-clipped: bases in 5' and 3' of the read are NOT part of the alignment AND those bases have been removed from the read sequence in the BAM file 這部分沒比對(duì)上并且沒有保留在SAM/BAM比對(duì)結(jié)果中
# 還是上面??clipped alignment的例子： # 如果是：3H4M1D5M3H，那么保留在SAM中的結(jié)果就是：TTGCTCCGCC建丧，而不是CGATTGCTCCGCCAGG（雖然CGA沒有比對(duì)上，但依然在soft clipped結(jié)果中保留下來） Read: TTGC-TCCGCC | |||| |||||| Ref:CCGATCGAGACTTGCGTCCGCCTCCCGATCA
- soft-clipped: if your cigar is
10S10M10Sthen the SEQ and base-quals will be 30 bases long. 利用soft-clipping算法可以避開由于read兩側(cè)質(zhì)量低而導(dǎo)致整條read比對(duì)不上的現(xiàn)象橄维。不過即使被標(biāo)記為soft clipped保留下來尺铣，依然不會(huì)在找變異争舞、基因組瀏覽器可視化凛忿、計(jì)算覆蓋度等過程中被使用（例如https://www.biostars.org/p/255062/中就提到：在計(jì)算基因組覆蓋度時(shí)竞川，幾乎全部工具bamCoverage/multiBamSummary甚至samtools view -c都會(huì)忽略soft clip情況） - hard-clipped: if your cigar is:
10H10M10Hthen the SEQ will only be 10 bases long.
那么什么時(shí)候標(biāo)記Hard clip，什么時(shí)候標(biāo)記Soft clip呢委乌？
參考：https://www.biostars.org/p/310722/ 和 https://www.biostars.org/p/109333/
in bwa, HARD clipping is used for supplementary reads.
if the read has a chimeric alignment, the paired
or the top hit uses soft clipping ；All the other hits part of the chimeric alignment will use hard clippingchimeric alignment: “嵌合比對(duì)” 的形成是由于一條測(cè)序read比對(duì)到基因組上時(shí)分別比對(duì)到兩個(gè)不同的區(qū)域遭贸，而這兩個(gè)區(qū)域基本沒有overlap戈咳。因此它在sam文件中需要占用多行記錄顯示。只有第一個(gè)記錄被稱作"representative",其他的都是"supplementary"【Chimeric reads are also called split reads】革砸；RNA-seq中的chimeric read或許可以說明有融合基因存在除秀，但在基因組中一般作為結(jié)構(gòu)變異的證據(jù)
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