Pilon step runs out of error (java.lang.OutOfMemoryError)

[RaverJay]

Hey,

a unicycler run threw the following error on the pilon step:

Polishing assembly with Pilon (2018-10-12 15:50:32)
Unicycler now conducts multiple rounds of Pilon in an attempt to repair any remaining small-
scale errors with the assembly.

Aligning reads to find appropriate insert size range...
Insert size 1st percentile:  134
Insert size 99th percentile: 567

Pilon polish round 1
Total number of changes: 40

Pilon polish round 2
Total number of changes: 12

Pilon polish round 3
Total number of changes: 9

Pilon polish round 4
Total number of changes: 2

Pilon polish round 5
No Pilon changes

Pilon polish round 6
Unable to polish assembly using Pilon: Pilon encountered an error:
Pilon version 1.22 Wed Mar 15 16:38:30 2017 -0400
Genome: 6_polish_input.fasta
Fixing snps, indels, gaps, local
Input genome size: 975500
Scanning BAMs
6_paired_alignments.bam: 16290524 reads, 0 filtered, 15509205 mapped, 14678436 proper, 789568 stray, FR 100% 278+/-86, max 536
Processing 3:1-12069
frags 6_paired_alignments.bam: coverage 3330
Total Reads: 418549, Coverage: 3330, minDepth: 333
Confirmed 12067 of 12069 bases (99.98%)
Corrected 0 snps; 0 ambiguous bases; corrected 0 small insertions totaling 0 bases, 0 small deletions totaling 0 bases
# Attempting to fix local continuity breaks
# fix break: 3:11297-11602 0 -0 +0 NoSolution TandemRepeat 48
3:1-12069 log:
Finished processing 3:1-12069
Processing 1:1-718813
frags 6_paired_alignments.bam: coverage 1511
Total Reads: 10898551, Coverage: 1511, minDepth: 151
Confirmed 718573 of 718813 bases (99.97%)
Corrected 0 snps; 0 ambiguous bases; corrected 0 small insertions totaling 0 bases, 0 small deletions totaling 0 bases
# Attempting to fix local continuity breaks
# fix break: 1:1586-1932 0 -0 +0 NoSolution TandemRepeat 30
Exception in thread "main" java.lang.reflect.InvocationTargetException
	at sun.reflect.NativeMethodAccessorImpl.invoke0(Native Method)
	at sun.reflect.NativeMethodAccessorImpl.invoke(NativeMethodAccessorImpl.java:62)
	at sun.reflect.DelegatingMethodAccessorImpl.invoke(DelegatingMethodAccessorImpl.java:43)
	at java.lang.reflect.Method.invoke(Method.java:498)
	at com.simontuffs.onejar.Boot.run(Boot.java:340)
	at com.simontuffs.onejar.Boot.main(Boot.java:166)
Caused by: java.lang.OutOfMemoryError: GC overhead limit exceeded
	at java.util.Arrays.copyOfRange(Arrays.java:3664)
	at java.lang.String.<init>(String.java:207)
	at java.lang.String.substring(String.java:1969)
	at scala.collection.immutable.StringOps$.slice$extension(StringOps.scala:44)
	at org.broadinstitute.pilon.Assembler$$anonfun$addToPileups$1.apply$mcVI$sp(Assembler.scala:82)
	at scala.collection.immutable.Range.foreach$mVc$sp(Range.scala:160)
	at org.broadinstitute.pilon.Assembler.addToPileups(Assembler.scala:81)
	at org.broadinstitute.pilon.Assembler.addRead(Assembler.scala:70)
	at org.broadinstitute.pilon.Assembler$$anonfun$addReads$1.apply(Assembler.scala:48)
	at org.broadinstitute.pilon.Assembler$$anonfun$addReads$1.apply(Assembler.scala:48)
	at scala.collection.immutable.List.foreach(List.scala:381)
	at org.broadinstitute.pilon.Assembler.addReads(Assembler.scala:48)
	at org.broadinstitute.pilon.GapFiller.assembleIntoBreak(GapFiller.scala:124)
	at org.broadinstitute.pilon.GapFiller.assembleAcrossBreak(GapFiller.scala:52)
	at org.broadinstitute.pilon.GapFiller.fixBreak(GapFiller.scala:45)
	at org.broadinstitute.pilon.GenomeRegion$$anonfun$identifyAndFixIssues$4.apply(GenomeRegion.scala:383)
	at org.broadinstitute.pilon.GenomeRegion$$anonfun$identifyAndFixIssues$4.apply(GenomeRegion.scala:381)
	at scala.collection.immutable.List.foreach(List.scala:381)
	at org.broadinstitute.pilon.GenomeRegion.identifyAndFixIssues(GenomeRegion.scala:381)
	at org.broadinstitute.pilon.GenomeFile$$anonfun$processRegions$4.apply(GenomeFile.scala:119)
	at org.broadinstitute.pilon.GenomeFile$$anonfun$processRegions$4.apply(GenomeFile.scala:108)
	at scala.collection.Iterator$class.foreach(Iterator.scala:893)
	at scala.collection.AbstractIterator.foreach(Iterator.scala:1336)
	at scala.collection.parallel.ParIterableLike$Foreach.leaf(ParIterableLike.scala:972)
	at scala.collection.parallel.Task$$anonfun$tryLeaf$1.apply$mcV$sp(Tasks.scala:49)
	at scala.collection.parallel.Task$$anonfun$tryLeaf$1.apply(Tasks.scala:48)
	at scala.collection.parallel.Task$$anonfun$tryLeaf$1.apply(Tasks.scala:48)
	at scala.collection.parallel.Task$class.tryLeaf(Tasks.scala:51)
	at scala.collection.parallel.ParIterableLike$Foreach.tryLeaf(ParIterableLike.scala:969)
	at scala.collection.parallel.AdaptiveWorkStealingTasks$WrappedTask$class.compute(Tasks.scala:152)
	at scala.collection.parallel.AdaptiveWorkStealingForkJoinTasks$WrappedTask.compute(Tasks.scala:443)
	at scala.concurrent.forkjoin.RecursiveAction.exec(RecursiveAction.java:160)

Two things are strange here: The previous round of pilon didnt change anything, so the crashing round seems superfluous.
Also, unicycler does not seem to care and gives:

Assembly complete (2018-10-12 19:47:02)
Saving /data/mahlzeitlocal/sebastian/mycoplasma_bovis_asm/unicycler/15DD0163/assembly.gfa
Saving /data/mahlzeitlocal/sebastian/mycoplasma_bovis_asm/unicycler/15DD0163/assembly.fasta


real	269m19.594s
user	2189m14.516s
sys	10m13.884s
>>> Done.
>>> All done.

So I would expect it is indeed finished - still seems like a bug or some incorrect memory allocation for pilon.

[apredeus]

I've always changed the settings of memory that Pilon is allowed to use, otherwise it crashes quite often.

Just find pilon wrapper and change this line

default_jvm_mem_opts = ['-Xms512m', '-Xmx1g']

to

default_jvm_mem_opts = ['-Xms512m', '-Xmx8g']

or something appropriate for your system. I think 8 Gb should be enough for any bacterial genome.

[RaverJay]

True, seems rather obvious that 1GiB is too low. Thanks!

For anyone seeing this and wondering about where to change the line:
The pilon executable (e.g. from conda) is just a python wrapper script to run the java program.

e.g. /home/<user>/miniconda3/bin/pilon