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I am trying to use Pilon to improve a reference based on some Illumina data I have got. So, I aligned the Illumina reads to the reference using bwa. Then, I want to use Pilon to improve the reference using the alignment file in BAM format.

Information about Pilon is found here. I installed Pilon using conda.

The output of 'pilon' is:

Pilon version 1.22 Wed Mar 15 16:38:30 2017 -0400

    Usage: pilon --genome genome.fasta [--frags frags.bam] [--jumps jumps.bam] [--unpaired unpaired.bam]
                 [...other options...]
           pilon --help for option details

The output of:

pilon --genome ref.fa --frags readsSorted.bam --output pilo --outdir output/scaffold

is:

 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: Java heap space
    at org.broadinstitute.pilon.GenomeRegion.<init>(GenomeRegion.scala:55)
    at org.broadinstitute.pilon.GenomeFile$$anonfun$contigRegions$1.apply(GenomeFile.scala:72)
	at org.broadinstitute.pilon.GenomeFile$$anonfun$contigRegions$1.apply(GenomeFile.scala:72)
    at scala.collection.TraversableLike$$anonfun$map$1.apply(TraversableLike.scala:234)
	at scala.collection.TraversableLike$$anonfun$map$1.apply(TraversableLike.scala:234)
    at scala.collection.immutable.Range.foreach(Range.scala:160)
    at scala.collection.TraversableLike$class.map(TraversableLike.scala:234)
	at scala.collection.AbstractTraversable.map(Traversable.scala:104)
	at org.broadinstitute.pilon.GenomeFile.contigRegions(GenomeFile.scala:72)
	at org.broadinstitute.pilon.GenomeFile$$anonfun$2.apply(GenomeFile.scala:52)
    at org.broadinstitute.pilon.GenomeFile$$anonfun$2.apply(GenomeFile.scala:52)
    at scala.collection.immutable.List.map(List.scala:277)
    at org.broadinstitute.pilon.GenomeFile.<init>(GenomeFile.scala:52)
    at org.broadinstitute.pilon.Pilon$.main(Pilon.scala:98)
    at org.broadinstitute.pilon.Pilon.main(Pilon.scala)
    ... 6 more
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    $\begingroup$ Note that the error is a memory issue: java.lang.OutOfMemoryError: Java heap space $\endgroup$
    – gringer
    Nov 16 '17 at 22:12
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Like gringer already commented, the cause of your error is nicely given by the java program. Your default max heap settings are probably too low for the program. If you have more memory on your machine you can give max heap a higher value. See this post and answers on Stack overflow.

You'll probably need to use the -Xmxn argument in java.

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"If pilon was installed by condo, edit the file "/Users/user/anaconda/bin/pilon" by changing the maximum memory limit from 1G to higher numbers like "14G". It works. " check the thread: https://github.com/broadinstitute/pilon/issues/26

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