Hi all I been testing a paired RNA.seq sample. I start with a paired of FASTQ files and do alignment with STAR and run STAR-Fusion. Everything looks good until I try to reverse the BAM files generated from STAR originally to fastq and rerun STAR-Fusion; this resulted in about half the fusions that as originally called. So making a mistake somewhere when converting from BAM to fastq. So far I tried several tools including samtools fastq, picard RevertSam to a ubam and then SamToFastq, I also tried bamtofastq but nothing works. Here is what I did with the original STAR alingment.
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 Bam looks like.
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)
Here is what I tried with samtools.
samtools sort -n --threads 4 ./T1Aligned.sortedByCoord.out.bam|samtools fastq -1 T1_R1_.fq -2 T1_R2_.fq -0 test.fq -
/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 bamtofastq but nothing seem to be able to convert the bam back to where I was able to call the fusions correctly. Any suggestions? Thanks!