MiXCR java.lang.NullPointerException

[weshorton]

Overview

While working on issue #11, mixcr began to throw an error while trying to maintain read IDs during the align and assembly process. We need to include the read ids in our pipeline so that we can map back to original reads to figure out what's happening to them in the align/assemble steps that's causing us to lose so many. Below is a copy of the issue that I created on the MiXCR github page

System

java version 1.7.0_101 mixcr version 1.7.1

Commands

usr/bin/java -Xmx15g -jar /path/to/mixcr-1.7.1/mixcr.jar align -f --loci TRB --species mmu --save-description --save-reads --report /path/to/report.txt /path/to/fastq /path/to/align.vdjca

usr/bin/java -Xmx15g -jar /path/to/mixcr-1.7.1/mixcr.jar assemble -f --index /path/to/index.txt --report /path/to/report.txt --threads 4 /path/to/align.vdjca /path/to/assemble.clns

Description

I'm running the mixcr align-assemble-export pipeline on 170 samples of mouse T-Cell Receptor beta data. Samples were created using a multiplex PCR reaction to amplify all 20 V and 13 J genes within this locus. Using the pipeline without the index step works just fine for all samples, it's when we attempt to add the -index [index file] option that we get the issue.

The error occurs for 8 of the 170 files, but which 8 vary between runs.

Examples

Trial 1

1. Run mixcr align (no errors) and mixcr assemble

   A) Receive two errors from assemble (Samples 146 and 167)

2. Using same vdjca files, re-run mixcr assemble for samples 146 and 167

   A) Did this 10 times for each sample

    i) receive same error message 10 of 10 times for sample 146
    ii) receive same error message 10 of 10 times for sample 167

   Trial 2

3. Run mixcr align (no errors) and mixcr assemble

   A) Receive four errors from assemble (Samples 1, 29, 86, 95, 145, 167)

4. Using the same vdjca files, re-run mixcr assemble for samples 1, 29, 86, 95, 145, 167

   A) Did this 10 times for each sample - receive same error message 10 of 10 times for all 6

   Trial 3

5. Run mixcr align (no errors) and mixcr assemble A) Receive three errors from assemble (Samples 95, 146, 167)
6. Re-run mixcr align for error samples 10 times

7. Re-run mixcr assemble for the outputs of part 2.

   A) For the 10 replicates of sample 95 - 5 produced error B) For the 10 replicates of sample 146 - 3 produced error C) For the 10 replicates of sample 167 - 7 produced error

   Stack Trace of Error

Exception in thread "main" java.lang.NullPointerException at com.milaboratory.mixcr.assembler.IO.write0(IO.java:122) at com.milaboratory.mixcr.assembler.IO.access$000(IO.java:45) at com.milaboratory.mixcr.assembler.IO$ReadToCloneMappingBtreeSerializer.serialize(IO.java:81) at org.mapdb.BTreeMap$NodeSerializer.serialize(BTreeMap.java:385) at org.mapdb.BTreeMap$NodeSerializer.serialize(BTreeMap.java:288) at org.mapdb.Store.serialize(Store.java:154) at org.mapdb.StoreDirect.put(StoreDirect.java:365) at org.mapdb.Caches$HashTable.put(Caches.java:216) at org.mapdb.Pump.buildTreeMap(Pump.java:470) at org.mapdb.DB.createTreeSet(DB.java:1072) at org.mapdb.DB$BTreeSetMaker.make(DB.java:749) at com.milaboratory.mixcr.cli.ActionAssemble.go(ActionAssemble.java:125) at com.milaboratory.mitools.cli.JCommanderBasedMain.main(JCommanderBasedMain.java:145) at com.milaboratory.mixcr.cli.Main.main(Main.java:64)

Interpretation

To me it looks like there is an issue with saving reads in the align step, but one that does not trigger any error. It only shows when the assemble step is run and tries map the read IDs into the index file.

Next Steps

Going to take one of the problem files (167) and divide the fastq file up into 10 segments of relatively equal size and perform the same trials as above.

[leyshock]

Per MiXCR development team, bug should be fixed in 1.7.4 release, week of ~6 June.

[weshorton]

Receiving same error with mixcr v.1.8.1 - sent message to developers

[weshorton]

To Do

1. Leave this as a "labeled" issue with no milestone. label is #4 "technical"