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BAM -> FASTQ why can I not produce the same result?

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  • BAM -> FASTQ why can I not produce the same result?

    Dear all, I've been trying to test if I can produce the same fusion call starting from BAM instead of FASQ. My STAR command is as such.

    HTML Code:
    STAR --genomeDir /index/hg38.p5/ \
    --readFilesIn T1_1.fq.gz T1_2.fq.gz \
    --readFilesCommand zcat --outFileNamePrefix T1 --outSAMtype BAM SortedByCoordinate --quantMode GeneCounts --sjdbGTFfile /index/hg38.gtf
    This is how the resulting BAM file looks like.

    HTML Code:
    samtools flagstat T1Aligned.sortedByCoord.out.bam
    
    121206267 + 0 in total (QC-passed reads + QC-failed reads)
    11756519 + 0 secondary
    0 + 0 supplementary
    0 + 0 duplicates
    121206267 + 0 mapped (100.00% : N/A)
    109449748 + 0 paired in sequencing
    54766664 + 0 read1
    54683084 + 0 read2
    109354332 + 0 properly paired (99.91% : N/A)
    109354332 + 0 with itself and mate mapped
    95416 + 0 singletons (0.09% : N/A)
    0 + 0 with mate mapped to a different chr
    0 + 0 with mate mapped to a different chr (mapQ>=5)
    However, I have tried a number of tools including the following.
    HTML Code:
    1. samtools sort -n --threads 4 ./T1Aligned.sortedByCoord.out.bam|samtools fastq -1 T1_R1_.fq -2 T1_R2_.fq -0 test.fq -
    I tried converting back to uBAM and doing it again.

    HTML Code:
    /picard-tools-2.2.4/picard.jar RevertSam I=T1Aligned.sortedByCoord.out.bam O=T1.ubam QUIET=true VALIDATION_STRINGENCY=SILENT
    
    /picard-tools-2.2.4/picard.jar SamToFastq INPUT=T1.ubam FASTQ=T1_R1_.fq SECOND_END_FASTQ=T2_R2_.fq VALIDATION_STRINGENCY=SILENT
    I also tried out this tutorial herehttps://gist.github.com/darencard/72...64ca512a04a6dd, but failed because of some read mismatch.

    I also tried bedtools bamtofastq but nothing works. When I input fastq vs what I can derive from BAM-> Fastq I'm not getting the same fusion output as I did previously. Anybody with suggestions? Thanks!

  • #2
    Cross-posted on Biostars

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