BAM file is empty or no read alignments are located in exonic regions.

[Gabriel Gama]

When doing RNAseqQC, from STAR aligned transcriptome sequencing, i'm getting:

code:

qualimap --java-mem-size=24G rnaseq -bam {1} -gtf /home/references/ENSEMBL/star/Homo_sapiens.GRCh38.103.chr_patch_hapl_scaff.gtf -oc /home//qualimap/RNAseqQC/counts/{1/.} -p non-strand-specific -pe -outdir /home/qualimap/RNAseqQC -outfile {1/.}_RNAseqQC_ensembl.pdf

result:

WARNING! Number of non-correct SAM format alignments skipped: 155683433

Which is 100% of the reads. This results in:

BAM file is empty or no read alignments are located in exonic regions.

I aligned with star (with extra commands for star-fusion): 

STAR --genomeDir /home/references/ENSEMBL/star/ --readFilesIn {1} {2} --outSAMtype BAM SortedByCoordinate --outReadsUnmapped None --twopassMode Basic --outSAMstrandField intronMotif --outSAMunmapped Within --chimSegmentMin 12  --chimJunctionOverhangMin 8 --chimOutJunctionFormat 1  --alignSJDBoverhangMin 10 --alignMatesGapMax 100000 --alignIntronMax 100000 --alignSJstitchMismatchNmax 5 -1 5 5 --outSAMattrRGline ID:GRPundef --chimMultimapScoreRange 3 --chimScoreJunctionNonGTAG -4 --chimMultimapNmax 20 --chimNonchimScoreDropMin 10 --peOverlapNbasesMin 12 --peOverlapMMp 0.1 --alignInsertionFlush Right --alignSplicedMateMapLminOverLmate 0 --alignSplicedMateMapLmin 30 --outFileNamePrefix /home/star/{1/.}  --runThreadN 8 --outFilterScoreMinOverLread 0 --outFilterMatchNminOverLread 0 --outFilterMatchNmin 40

The line  --outSAMattrRGline ID:GRPundef generates a tag in the header that cannot be decoded by qualimap. So I adjusted the BAM files as suggested with:

samtools view -h {1} | grep -v "^@RG" > /home/samtools/{1/.}_rgfiltered.bam

--

Atenciosamente,

Gabriel A. C. Gama
Universidade Federal de São Paulo - UNIFESP
Laboratório Bases Genéticas dos Tumores da Tiroide
Rua Pedro de Toledo, 669 - 11º andar Vila Clementino São Paulo/SP - 04039-032
(11) 5576-4979 / (11) 999731226