Bam sort问题

Bam sort问题

本文作者：Sunny-King

发布时间：2022-07-22

本文链接：https://www.cnblogs.com/Sunny-King/p/Bioinformatics-Bam_sort.html

将两个bam文件合并到一块后发现Tag标签顺序不一致，回顾发现排序软件在排序时会对Tag的顺序做调整，并且不同软件之间也不一致。

一、问题

最近观察Bam文件时偶然发现之前从同一个文件提取出的bam文件，merge之后的Tag出现了顺序不一致的情况，经过排查发现其中一个bam经过了排序之后bam文件中Tag的顺序会与之前不一致。
为了验证是否是由于排序软件造成的，分别尝试了picard和samtools sort对同一个bam排序之后的tag顺序变化。

二、测试过程

1、测试参数

#Samtools
samtools sort test.bam -o test.samtools_sort.bam

# Picard
java ${JAVA_OPTS} -jar ${PICARD} SortSam \
    I=test.bam \
    O=test.picard_sort.bam \
    SORT_ORDER=coordinate

2、结果

从中随机挑出两条序列查看
输入序列

UMI-TAATA-TGCCT-5427_1        177        chr1        5531170        60        93M        chr9        115986372        0        CACCAGCAGACACGCGGCTGGACCAGGATTTGAGGCAAGCTGCAGCATTTCCTCCTGGTGCTGTTAGTGGTCTTCCCAGTAAGGAGTCTACAA        tlttprmttttpbtrttutusmsUttttrtmtnttttqnrmtstttkttiotptlttttttqtttttttttttltrttuttsqtttrtttsts        NM:i:0        MD:Z:93        MC:Z:93M        AS:i:93        XS:i:21        RG:Z:lib0225        BD:Z:jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjN        BI:Z:llllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllN        MI:Z:TAATA-TGCCT-5427        RX:Z:TAATA-TGCCT        XR:Z:TAATA-TGCCT-0        XZ:Z:1,2
UMI-ATAAT-GTGTT-32_1        145        chr1        3213457        60        29S35M        chr7        55086650        0        GCGAGCTAGACGTCCGGGCAGCCCCCGGCGCAGCACGGCTTAGCCTCACCCACCTTAACGCGCT        F-FEF=FEEFDEEFBDDFCEECEFEFDDDDDDDjZnmimljmkmmTjnnlmnnmlllmhlklnl        NM:i:0        MD:Z:35        MC:Z:93M   AS:i:35        XS:i:21        RG:Z:lib0225        SA:Z:chr7,55086703,-,34M30S,60,0;        BD:Z:HHHHHHHHHHHHHHHHHHHHHHHHHHHHHEEEEiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiN        BI:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjN        MI:Z:ATAAT-GTGTT-32        RX:Z:ATAAT-GTGTT   XR:Z:ATAAT-GTGTT-0        XZ:Z:1,1

Samtools sort输出序列

UMI-TAATA-TGCCT-5427_1        177        chr1        5531170        60        93M        chr9        115986372        0        CACCAGCAGACACGCGGCTGGACCAGGATTTGAGGCAAGCTGCAGCATTTCCTCCTGGTGCTGTTAGTGGTCTTCCCAGTAAGGAGTCTACAA        tlttprmttttpbtrttutusmsUttttrtmtnttttqnrmtstttkttiotptlttttttqtttttttttttltrttuttsqtttrtttsts        NM:i:0        MD:Z:93        MC:Z:93M        AS:i:93        XS:i:21        BD:Z:jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjN        BI:Z:llllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllN        MI:Z:TAATA-TGCCT-5427        RX:Z:TAATA-TGCCT        XR:Z:TAATA-TGCCT-0        XZ:Z:1,2        RG:Z:lib0225
UMI-ATAAT-GTGTT-32_1        145        chr1        3213457        60        29S35M        chr7        55086650        0        GCGAGCTAGACGTCCGGGCAGCCCCCGGCGCAGCACGGCTTAGCCTCACCCACCTTAACGCGCT        F-FEF=FEEFDEEFBDDFCEECEFEFDDDDDDDjZnmimljmkmmTjnnlmnnmlllmhlklnl        NM:i:0        MD:Z:35        MC:Z:93M   AS:i:35        XS:i:21        SA:Z:chr7,55086703,-,34M30S,60,0;        BD:Z:HHHHHHHHHHHHHHHHHHHHHHHHHHHHHEEEEiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiN        BI:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjN        MI:Z:ATAAT-GTGTT-32        RX:Z:ATAAT-GTGTT        XR:Z:ATAAT-GTGTT-0        XZ:Z:1,1        RG:Z:lib0225

Picard SortSam输出序列

UMI-TAATA-TGCCT-5427_1        177        chr1        5531170        60        93M        chr9        115986372        0        CACCAGCAGACACGCGGCTGGACCAGGATTTGAGGCAAGCTGCAGCATTTCCTCCTGGTGCTGTTAGTGGTCTTCCCAGTAAGGAGTCTACAA        tlttprmttttpbtrttutusmsUttttrtmtnttttqnrmtstttkttiotptlttttttqtttttttttttltrttuttsqtttrtttsts        MC:Z:93M        BD:Z:jjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjN        MD:Z:93        RG:Z:lib0225        BI:Z:llllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllllN        MI:Z:TAATA-TGCCT-5427        NM:i:0        XR:Z:TAATA-TGCCT-0        AS:i:93        XS:i:21        RX:Z:TAATA-TGCCT        XZ:Z:1,2
UMI-ATAAT-GTGTT-32_1        145        chr1        3213457        60        29S35M        chr7        55086650        0        GCGAGCTAGACGTCCGGGCAGCCCCCGGCGCAGCACGGCTTAGCCTCACCCACCTTAACGCGCT        F-FEF=FEEFDEEFBDDFCEECEFEFDDDDDDDjZnmimljmkmmTjnnlmnnmlllmhlklnl        SA:Z:chr7,55086703,-,34M30S,60,0;        MC:Z:93M        BD:Z:HHHHHHHHHHHHHHHHHHHHHHHHHHHHHEEEEiiiiiiiiiiiiiiiiiiiiiiiiiiiiiiN        MD:Z:35        RG:Z:lib0225        BI:Z:FFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFFjjjjjjjjjjjjjjjjjjjjjjjjjjjjjjN        MI:Z:ATAAT-GTGTT-32        NM:i:0        XR:Z:ATAAT-GTGTT-0        AS:i:35        XS:i:21        RX:Z:ATAAT-GTGTT        XZ:Z:1,1

三、结论

• Picard SortSam排序之后的Tag顺序与原始bam不一致
• Samtools sort排序之后的Tag顺序与原始bam不一致
• Picard SortSam和samtools sort排序之后的bam文件里不同read中Tag的顺序可能会有差异（有些reads可能会出现特有的Tag造成顺序异常）