BAM file is empty

[Samuel Clamons]

I've been trying to run qualimap rnaseq v2.1.3 on STAR-aligned transcriptome sequencing, and I'm getting a "BAM file is empty" error. I'm running with the command

qualimap rnaseq       -outdir ./          -oc counts.txt          -a proportional          -bam [filename]          -gtf mm10.gtf       --java-mem-size=16G

The GTF file is the same one used to build the STAR index (and to run STAR).

Output looks like:

Initializing regions from mm10.gtf...

Initialized 100000 regions...

Initialized 200000 regions...

Initialized 300000 regions...

Initialized 400000 regions...

Initialized 500000 regions...

Initialized 600000 regions...

Initialized 700000 regions...

Initialized 800000 regions...

Initialized 900000 regions...

Initialized 1000000 regions...

Initialized 1100000 regions...

Initialized 1128047 regions it total

Starting constructing transcripts for RNA-seq stats...

Finished constructing transcripts

Starting BAM file analysis

WARNING! The following chromosomes from reads are not found in annotations:

ENSMUST00000043951

ENSMUST00000150576

ENSMUST00000174266

ENSMUST00000153128

ENSMUST00000049932

ENSMUST00000128641

ENSMUST00000179954

ENSMUST00000072079

ENSMUST00000164181

ENSMUST00000111961

ENSMUST00000111962

ENSMUST00000111964

ENSMUST00000141217

ENSMUST00000027112

ENSMUST00000027036

ENSMUST00000056946

ENSMUST00000153892

ENSMUST00000178079

ENSMUST00000027071

ENSMUST00000085546

ENSMUST00000027271

ENSMUST00000087366

ENSMUST00000049385

ENSMUST00000121185

ENSMUST00000042235

ENSMUST00000147233

ENSMUST00000132618

ENSMUST00000046875

ENSMUST00000071732

ENSMUST00000152521

ENSMUST00000027338

ENSMUST00000075853

ENSMUST00000115538

ENSMUST00000139649

ENSMUST00000098994

ENSMUST00000129380

ENSMUST00000181382

ENSMUST00000027068

ENSMUST00000027343

ENSMUST00000140597

ENSMUST00000131177

ENSMUST00000021084

ENSMUST00000028592

ENSMUST00000155086

ENSMUST00000148476

ENSMUST00000097713

ENSMUST00000180722

ENSMUST00000149566

ENSMUST00000181973

ENSMUST00000093196

ENSMUST00000131511

ENSMUST00000173999

ENSMUST00000091130

ENSMUST00000134628

ENSMUST00000156816

ENSMUST00000111110

ENSMUST00000082414

ENSMUST00000027214

ENSMUST00000139443

ENSMUST00000163300

ENSMUST00000115352

ENSMUST00000165720

ENSMUST00000182984

ENSMUST00000086535

ENSMUST00000027050

ENSMUST00000082411

ENSMUST00000163749

ENSMUST00000088313

ENSMUST00000092511

ENSMUST00000171164

ENSMUST00000125005

ENSMUST00000151017

ENSMUST00000118155

ENSMUST00000131266

ENSMUST00000110505

ENSMUST00000082409

ENSMUST00000162031

ENSMUST00000072243

ENSMUST00000053266

ENSMUST00000058437

ENSMUST00000075242

ENSMUST00000082402

ENSMUST00000117589

ENSMUST00000114155

ENSMUST00000082408

ENSMUST00000027368

ENSMUST00000117146

ENSMUST00000059802

ENSMUST00000082405

ENSMUST00000153681

ENSMUST00000061242

ENSMUST00000140857

ENSMUST00000013845

ENSMUST00000081551

Failed to run rnaseq

java.lang.RuntimeException: BAM file is empty.

   at org.bioinfo.ngs.qc.qualimap.process.ComputeCountsTask.run(ComputeCountsTask.java:491)

   at org.bioinfo.ngs.qc.qualimap.process.RNASeqQCAnalysis.run(RNASeqQCAnalysis.java:68)

   at org.bioinfo.ngs.qc.qualimap.main.RnaSeqQcTool.execute(RnaSeqQcTool.java:188)

   at org.bioinfo.ngs.qc.qualimap.main.NgsSmartTool.run(NgsSmartTool.java:187)

    at org.bioinfo.ngs.qc.qualimap.main.NgsSmartMain.main(NgsSmartMain.java:111)

I've tried re-generating the STAR index to double-check that the GTF file is the right one. I've also tried changing one of the reads from mapping to an ENSMUST ID to the equivalent ENSMUSG ID, in case STAR wanted to use the former but qualimap the latter. Also tried sorting the BAM—no change. 

Any ideas what might be causing this?

[Samuel Clamons]

Never mind, a colleague figured it out—I was using the wrong output from STAR.