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Old 12-11-2018, 05:18 AM   #1
Alex Lee
Junior Member
 
Location: N. Cal

Join Date: Apr 2014
Posts: 10
Post 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.

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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.

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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.
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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.

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/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!
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Old 12-11-2018, 09:39 AM   #2
atpoint
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Location: Muenster

Join Date: Apr 2016
Posts: 13
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Cross-posted on Biostars
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bam conversion, fastq fasta reads, picard, samtools, star aligner

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