rsem-sam-validator

[Xiaofeng Qian]

I want to make the alignment file (.bam) satisfying RSEM's requirements.

But when I using this command, I met an error.

Here is the log file， is anyone can help me?

Here is the log file,

samtools sort -n -@ 1 -m 1G -o PA_5.AddRG.Reorder.convert_forRSEM.tmp.bam /storage2/workspace/RNAseq_SED_vs_PA/data/F23A430002092_MUSfxwlT/Alignment_hisat/bam/PA_5.AddRG.Reorder.Sort.bam
[bam_sort_core] merging from 13 files and 1 in-memory blocks...

rsem-scan-for-paired-end-reads 1 PA_5.AddRG.Reorder.convert_forRSEM.tmp.bam PA_5.AddRG.Reorder.convert_forRSEM.bam
.......................
Finished!

Conversion is completed. PA_5.AddRG.Reorder.convert_forRSEM.bam will be checked by 'rsem-sam-validator'.
rsem-sam-validator PA_5.AddRG.Reorder.convert_forRSEM.bam
.
The two mates of paired-end read V350220273L3C001R00100223784 are not adjacent!
The input file is not valid!

[Xiaofeng Qian]

this is my bam file

$samtools view -H PA_5.AddRG.Reorder.Sort.bam
@HD VN:1.6 SO:coordinate
@SQ SN:chr1 LN:195154279 M5:ebdcb59bd671b01de5a67d9803f500ea UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr10 LN:130530862 M5:cc1e9a05f16cacc4955f56bd0f579267 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr11 LN:121973369 M5:227171e89d468ee817a7fb0ccaba38cd UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr12 LN:120092757 M5:b0f91aa89b888e3c1f29263818b02210 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr13 LN:120883175 M5:fe93f607754e075f2849b35af77af07d UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr14 LN:125139656 M5:5587dd4c57372cc4fe78fe1f6f8b68d0 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr15 LN:104073951 M5:99f61c36433d8fc45bd265cffa4be357 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr16 LN:98008968 M5:8d8e220195ef6f4b400548600131d0d6 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr17 LN:95294699 M5:ca46671b6cafa344dddca2e6b0db0991 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr18 LN:90720763 M5:717a1f751989489983e41c7f195c202a UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr19 LN:61420004 M5:96b19db237dfb928d0695a3b366acf40 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr1_GL456210v1_random LN:169725 M5:0cc560d98f9f22f4385397db82e1c108 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr1_GL456211v1_random LN:241735 M5:36e85680c669756c9a1554cf31c9de03 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr1_GL456212v1_random LN:153618 M5:4c4dc3bfe987e3bc4ef4756bef269373 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr1_GL456221v1_random LN:206961 M5:e21da65d7276b256b8edf92660a928b0 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr1_MU069434v1_random LN:8412 M5:9de130f8b9ca81fa376dac13e6044f0b UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr1_GL456239v1_random LN:40056 M5:026a56195f744bfc337bd35c5c406ca3 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr2 LN:181755017 M5:08bc6a9988099d0d4cc457341933249a UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr3 LN:159745316 M5:2399e48fe19b768e0738efcedbc04b7a UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr4 LN:156860686 M5:c998850568fb66c8a0438a521965bf9a UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr4_JH584295v1_random LN:1976 M5:ebc2f8438cbd080b53dc1cf528bf070e UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr5 LN:151758149 M5:ded8e8ee2aae6a3e8c081e1a7c2e7c26 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr5_JH584296v1_random LN:199368 M5:9b5b5f3af54ac1c2e91964a2c8b3f9ee UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr5_JH584297v1_random LN:205776 M5:efb1b00ffad6dd710ffd5d46ce94a25c UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr5_JH584298v1_random LN:184189 M5:1910644b4393b414d16774d3a1b73c49 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr5_GL456354v1_random LN:195993 M5:61643b629b3105fd2f32cc82871ca8e0 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr5_JH584299v1_random LN:953012 M5:b6bc88bfe26ef155b5fe2a7b90830ca5 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr6 LN:149588044 M5:314397656eb3d25aaa70d407df67c6a8 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr7 LN:144995196 M5:cdc994b8172f4e1e3099a2b9a41f4684 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr7_GL456219v1_random LN:175968 M5:a9a02958a866bdbde864f45dba4678e9 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr8 LN:130127694 M5:33a6d892dcc698e5af446bd3cce98d61 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chr9 LN:124359700 M5:dce50387ea191577d2f47408be52d093 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrM LN:16299 M5:11c8af2a2528b25f2c080ab7da42edda UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456359v1 LN:22974 M5:603a636ed64b91e321f4016e95496e2e UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456360v1 LN:31704 M5:e920aab56a78dad48b143260fdd46def UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456366v1 LN:47073 M5:5a13a662666e5216edc9777123a90721 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456367v1 LN:42057 M5:c515676047df349605e1f0b9a2c931e2 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456368v1 LN:20208 M5:aafc72058b568c532611be3ed4de1421 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456370v1 LN:26764 M5:b1497fc4d9c50793ce42287b807023a6 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456372v1 LN:28664 M5:63d6c279d38036a35a614ebf3b5aff04 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456378v1 LN:31602 M5:f2e4730a3ebda87af4e300991b5b1fdd UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456379v1 LN:72385 M5:2e317d895c77ed23c1ead5d964787944 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456381v1 LN:25871 M5:cd52161045aaf547b16aa269806d0fa2 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456382v1 LN:23158 M5:0a6e2bdf8e33bbcf41a1f22d28b31040 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456383v1 LN:38659 M5:957a72650aef5448bb88d22c95091a3f UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456385v1 LN:35240 M5:14d999bf71d97ab109e0d120421813e1 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456387v1 LN:24685 M5:ee8d8a56353bfd1e03794413a6de4790 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456389v1 LN:28772 M5:33a8ae4e638372e6786e2139b868c9de UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456390v1 LN:24668 M5:98ab2165ef0892fb13b2826abef47f28 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456392v1 LN:23629 M5:4a6c8910adf1323d66f179e7b22c12b3 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456394v1 LN:24323 M5:d2fc329384f30769e5bb8e9e5547f88b UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_GL456396v1 LN:21240 M5:00bc9e8162a01525c883724d0f152c3a UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_JH584304v1 LN:114452 M5:3da6902c6bfae6e54184fe309460b5ce UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrUn_MU069435v1 LN:31129 M5:168e4d99aa1fa20c9740db4a2d0d4340 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrX LN:169476592 M5:2f96da7dbcddf6f0866de651f42417bb UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrX_GL456233v2_random LN:559103 M5:fea40855e95eb187dcbe56ae210d41dc UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrY LN:91455967 M5:493dcdf262cba21cae56d4092d5c9202 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrY_JH584300v1_random LN:182347 M5:63017c95f5328b37975b41ca1019ae14 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrY_JH584301v1_random LN:259875 M5:6b2fb727b7f71259d4b2aa53f921f83e UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrY_JH584302v1_random LN:155838 M5:8265ab6c29db6b3f7edbd3d0bf9f6649 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@SQ SN:chrY_JH584303v1_random LN:158099 M5:23d8b4c1dbfa3cc96e2e2d5f56914a31 UR:file:/ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic.fa
@RG ID:PA_5 LB:PA_5_library PL:illumina SM:PA_5 PU:machine
@PG ID:hisat2 PN:hisat2 VN:2.2.1 CL:"/ifswh4/BC_PUB_T1/Pipeline/Software/miniconda3/envs/hisat2/bin/hisat2-align-s --wrapper basic-0 --phred33 --sensitive --no-discordant --no-mixed -I 1 -X 1000 -p 8 -x /ifswh4/BC_PUB_T2/BioSysDB/BGI/v2201/Index/10090/UCSC/mm39/Index_geneome_Hisat2/genomic --novel-splicesite-outfile /ifswh7/BC_COM_RNA/F23A430002092/MUSfxwlT/result/backups/Alignment_hisat/PA_5/PA_5.junction -1 /tmp/65019.inpipe1 -2 /tmp/65019.inpipe2"
@PG ID:samtools PN:samtools PP:hisat2 VN:1.15.1 CL:/ifswh4/BC_PUB_T1/Pipeline/Software/miniconda3/envs/samtools/bin/samtools view -b -S -o /ifswh7/BC_COM_RNA/F23A430002092/MUSfxwlT/result/process/Alignment_hisat/PA_5/PA_5.bam -
@PG ID:samtools.1 PN:samtools PP:samtools VN:1.19.2 CL:samtools view -H PA_5.AddRG.Reorder.Sort.bam

Xiaofeng Qian <qianxiao...@gmail.com> 于2024年2月19日周一 00:01写道：

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