比对过程涉及的参数
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Soft-clipping
primary alignment
Secondary alignments
A secondary alignment occurs when a given read could align reasonably well to more than one place. One of the possible reported alignments is termed "primary" and the others will be marked as "secondary".Unmapped reads are in the BAM file but have no valid assigned position (N.B., they may have an assigned position, but it should be ignored). It's typically the case that a number of reads can't be aligned, due to things like sequencing errors, imperfect matches between the DNA sequenced and the reference, random e. coli or other contamination, etc..
supplemental alignments.
https://yulijia.net/en/bioinformatics/2015/12/21/Linear-Chimeric-Supplementary-Primary-and-Secondary-Alignments.html#fn:1
https://hongiiv.tistory.com/844 -
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https://www.cnblogs.com/timeisbiggestboss/p/8856888.htmlSecondary ,Supplementary alignment 和bwa mem的-M -Y参数
1.supplementary alignmentsupplementary alignment是指一条read的一部分和参考区域1比对成功，另一部分和参考区域2比对成功，参考区域1和参考区域2没有交集（或很少），那么一条read就会产生两条sam文件，
将其中的一条sam文件作为represent alignment，而另一条作为supplementary alignment （flag为2048）。
将上面的fastq文件去跑bwa，read有两条sam文件，第二条的flag值为2048：
2.bwa mem的-M -Y参数：
-M：mark shorter split hits as secondary。就是把supplemenary alignment 变为no primary（flag值256） 。下面是bwa mem -M的结果
-Y:use soft clipping for supplementary alignments。把默认的hard clip变为soft clip。hard clip 不会显示不匹配的碱基串，soft clip会显示不匹配的碱基串。下面是bwa mem -Y的结果（58H34M变为58S34M）
3.secondary(no primary)是指这条read在基因组上有多个匹配区域（>=2），可以是read是的同一部分有不同匹配区域，也可以是一条read上的不同区域。所以supplemenary aligment应该算是secondary的子集。
许多处理bam的软件不会去处理supplemenary（split alignments），比如Picard’s markDuplicates，所以可能需要用-M把supplemenary 转换为secondary。
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https://gist.github.com/crazyhottommy/ed73c7e2daee8383dccb35f224f99714
获取唯一比对的序列
samtools view -h my.bam | awk '$17 ~ /XA:/' || $1 ~ /^@/' | samtools view -bS - > my_unique.bam
