Closed bbimber closed 7 years ago
This seems to have something to do with absence of D genes in the library. Basically it looks like a bug.) I will now try to fix it and send you new jar, so you can confirm that it fixes the problem.
Please try this version, and let me know if it fixes the issue: http://files.milaboratory.com/mixcr/mixcr-2.0.3-hotfix.zip
It did fix the NPE. I'm going to try to add D genes to our library anyway, since this is probably a good idea. Thanks!
Great!
Hello,
We just updated to Mixcr 2.0.2, and are seeing an NPE when running assemblePartial. It seems to have something to do w/ either not lacking any TRB hits, or our custom library. See the commands, outputs and stack below:
run this
mixcr align -f -r log.txt -s rhesus -OallowPartialAlignments=true -g -p rna-seq --library rhesus -OallowChimeras=true -t 8 Clone39-R1.fastq.gz Clone39-R2.fastq.gz Clone39.1.rhesus.mixcr.vdjca
output below, note TRA matching only
Reference library: rhesus:9544 (6daa1e92265f95d206ebff3ff02cd2a8) WARNING: Functional gene TRBV3-1*01 doesn't contain full VTranscriptWithout5UTRWithP (excluded) WARNING: ... 63 more functional genes excluded due to absent "featureToAlign". Alignment: 100% ============= Report ============== Analysis time: 1.03s Total sequencing reads: 254 Successfully aligned reads: 42 (16.54%) Alignment failed, no hits (not TCR/IG?): 126 (49.61%) Alignment failed because of absence of CDR3 parts: 74 (29.13%) Alignment failed because of low total score: 12 (4.72%) Overlapped: 60 (23.62%) Overlapped and aligned: 10 (3.94%) Overlapped and not aligned: 50 (19.69%) [TRA] chains: 42 (100%)
then run this, which fails with the NPE
mixcr assemblePartial -p -f -r log.txt Clone39.1.rhesus.mixcr.vdjca Clone39.1.rhesus.mixcr.partial.1.vdjca Searching for overlaps: 100% Exception in thread "main" java.lang.NullPointerException at com.milaboratory.mixcr.partialassembler.PartialAlignmentsAssemblerAligner.process0(PartialAlignmentsAssemblerAligner.java:102) at com.milaboratory.mixcr.partialassembler.PartialAlignmentsAssemblerAligner.process0(PartialAlignmentsAssemblerAligner.java:26) at com.milaboratory.mixcr.vdjaligners.VDJCAligner.process(VDJCAligner.java:74) at com.milaboratory.mixcr.partialassembler.PartialAlignmentsAssembler.searchOverlaps(PartialAlignmentsAssembler.java:129) at com.milaboratory.mixcr.cli.ActionAssemblePartialAlignments.go(ActionAssemblePartialAlignments.java:51) at com.milaboratory.cli.JCommanderBasedMain.main(JCommanderBasedMain.java:147) at com.milaboratory.mixcr.cli.Main.main(Main.java:113)
As a test, I repeated the align command, except included "-OvParameters.geneFeatureToAlign=VRegion" in the align command:
mixcr align -f -OvParameters.geneFeatureToAlign=VRegion -r log.txt -s rhesus -OallowPartialAlignments=true -g -p rna-seq --library rhesus -OallowChimeras=true -t 8 Clone39-R1.fastq.gz Clone39-R2.fastq.gz Clone39.1.rhesus.mixcr.vdjca
output, seeing a TRB hit:
Alignment: 100% ============= Report ============== Analysis time: 1.34s Total sequencing reads: 254 Successfully aligned reads: 40 (15.75%) Alignment failed, no hits (not TCR/IG?): 133 (52.36%) Alignment failed because of absence of CDR3 parts: 70 (27.56%) Alignment failed because of low total score: 11 (4.33%) Overlapped: 60 (23.62%) Overlapped and aligned: 12 (4.72%) Overlapped and not aligned: 48 (18.9%) [TRA] chains: 39 (97.5%) [TRB] chains: 1 (2.5%)
Then if you run the exact same assemblePartial command as above, it now works.