Problem running CollectRnaSeqMetrics

Question: Problem running CollectRnaSeqMetrics

2.8 years ago by

United States

Hello,

I am trying to run Collectrnaseqmetrics in usegalaxy toolshed.g2.bx.psu.edu/repos/devteam/picard/picard_CollectRnaSeqMetrics/1.126.0 and am encountering an error (see below). Parameters are below. Can you help me troubleshoot?

Thanks!

Brian Hermann

Fatal error: Exit code 1 ()
Picked up _JAVA_OPTIONS: -Djava.io.tmpdir=/galaxy-repl/main/scratch
Exception in thread "main" picard.PicardException: Sequence dictionaries differ in /galaxy-repl/main/files/014/232/dataset_14232457.dat and /galaxy-repl/main/files/014/232/dataset_14232298.dat
	at picard.analysis.directed.RnaSeqMetricsCollector.makeOverlapDetector(RnaSeqMetricsCollector.java:76)
	at picard.analysis.CollectRnaSeqMetrics.setup(CollectRnaSeqMetrics.java:109)
	at picard.analysis.SinglePassSamProgram.makeItSo(SinglePassSamProgram.java:100)
	at picard.analysis.SinglePassSamProgram.doWork(SinglePassSamProgram.java:53)
	at picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:187)
	at picard.cmdline.PicardCommandLine.instanceMain(PicardCommandLine.java:89)
	at picard.cmdline.PicardCommandLine.main(PicardCommandLine.java:99)
Caused by: htsjdk.samtools.util.SequenceUtil$SequenceListsDifferException: Sequences at index 20 don't match: 20/227966/chr4_GL456350_random 20/14945/chr4_JH584292_random/UR=file:/space/jen/new_genome/mm10/picard_index/mm10.fa/M5=c2ff41899e0f684fd93b28c58756e02f
	at htsjdk.samtools.util.SequenceUtil.assertSequenceListsEqual(SequenceUtil.java:121)
	at htsjdk.samtools.util.SequenceUtil.assertSequenceDictionariesEqual(SequenceUtil.java:169)
	at picard.analysis.directed.RnaSeqMetricsCollector.makeOverlapDetector(RnaSeqMetricsCollector.java:74)
	... 6 more

Input Parameter	Value	Note for rerun
Select SAM/BAM dataset or dataset collection	16: TopHat on data 2 and data 1: accepted_hits
Load reference genome from	cached
Using reference genome	mm10
Gene annotations in refFlat form	197: mm10 refFlat
Location of rRNA sequences in genome, in interval_list format	199: mm10 rRNA interval list (UCSC)
What is the RNA-seq library strand specificity	None
When calculating coverage based values use only use transcripts of this length or greater	500
This percentage of the length of a fragment must overlap one of the ribosomal intervals for a read or read pair to be considered rRNA.	0.8
The level(s) at which to accumulate metrics	All reads
Assume the input file is already sorted	True
Select validation stringency	Lenient

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modified 2.8 years ago by Jennifer Hillman Jackson ♦ 25k • written 2.8 years ago by brian.hermann • 40

2.8 years ago by

Jennifer Hillman Jackson ♦ 25k

United States

Jennifer Hillman Jackson ♦ 25k wrote:

Hello,

These tools had previous issues that have not been confirmed to be completely resolved. OR the input may just need to be coordinate sorted (use Sort Sam).

https://github.com/jennaj/support-known-issues/wiki

A sort order conflict or some other data mismatch problem are the two potentials for the immediate problem. Once corrected, if an error results, then the original bug report error (dependency related) would come up. See link above for those details and follow it for the confirmation of a successful fix at usegalaxy.org.

Would you be able to share a bug report for testing? Replicate at http://usegalaxy.org (if that is not where you are working right now) and click on the green bug icon to send in the report. Please include a link to this Biostars post to help us associated the two.

If something else is going on, we will write back plus add those notes to the tickets tracking the issue with this tool (and another related tool).

Thanks, Jen, Galaxy team

ADD COMMENT • link written 2.8 years ago by Jennifer Hillman Jackson ♦ 25k

Thanks Jen.

Bug report issued. I have had this issue with multiple datasets that were previous analyzed successfully with this tool (using mm9, mm10, Hg19 genome annotations).

Best,

Brian

ADD REPLY • link written 2.8 years ago by brian.hermann • 40

Hi Brian,

There is some sort of dependency issue with the tool. As I mentioned in the direct email, the path to the reference genome is incorrect (and I missed that when reviewing the first time). An issue has been created to track the problem that you, plus any others that have the problem, can follow for the resolution. It is linked from here: https://github.com/jennaj/support-known-issues/wiki

Thanks again for sending in the bug report details, Jen, Galaxy team

ADD REPLY • link written 2.8 years ago by Jennifer Hillman Jackson ♦ 25k

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