-ProductionSNPCallerPluginV2, Length Test

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Stephanie

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May 10, 2018, 4:18:21 AM5/10/18

to TASSEL - Trait Analysis by Association, Evolution and Linkage

Hello,

I am attempting to analyse two paired-end datasets together, both with different read lengths: one is 100bp read lengths, the other is 150bp read lengths. I've been able to successfully do this in the past by demultiplexing the reads and re-appending barcodes before running through Tassel5 GBS v2 Pipeline with kmerLength specified to 64bp.

This time around, I wanted to trim the data before processing it with Tassel5. First, I trimmed the demultiplexed reads to remove the Illumina adapter and the cut site, and also ran a custom script to verify if any chimeras were present (see scripts here). Then, I trimmed all the reads down to 65bp using Skewer to make sure that both my data sets were the same size, to reduce batch effects.

Finally, I interleaved my forward/reverse reads using ShuffleSequences_fasta.pl (found in Velvet), and appended new ApeKI barcodes, generated with GBSX, using a custom script.

When I run these fastq files through Tassel5 GBS v2 Pipeline, I get through all plugins, except for -ProductionSNPCallerPluginV2, where I get the following output (see errors in red):

Memory Settings: -Xms512m -Xmx16G

Tassel Pipeline Arguments: -fork1 -ProductionSNPCallerPluginV2 -db /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/2016_2018_Kaki.db -e ApeKI -i /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018 -k /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/keyfile.barcoded.txt -kmerLength 64 -o /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/GBS_2016_2018_9May2018.vcf -endPlugin -runfork1

[main] INFO net.maizegenetics.tassel.TasselLogging - Tassel Version: 5.2.43 Date: February 22, 2018

[main] INFO net.maizegenetics.tassel.TasselLogging - Max Available Memory Reported by JVM: 14564 MB

[main] INFO net.maizegenetics.tassel.TasselLogging - Java Version: 1.8.0_162

[main] INFO net.maizegenetics.tassel.TasselLogging - OS: Linux

[main] INFO net.maizegenetics.tassel.TasselLogging - Number of Processors: 8

[main] INFO net.maizegenetics.pipeline.TasselPipeline - Tassel Pipeline Arguments: [-fork1, -ProductionSNPCallerPluginV2, -db, /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/2016_2018_Kaki.db, -e, ApeKI, -i, /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018, -k, /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/keyfile.barcoded.txt, -kmerLength, 64, -o, /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/GBS_2016_2018_9May2018.vcf, -endPlugin, -runfork1]

net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2

[pool-1-thread-1] INFO net.maizegenetics.plugindef.AbstractPlugin - Starting net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2: time: May 10, 2018 0:11:21

Enzyme: ApeKI

[pool-1-thread-1] INFO net.maizegenetics.plugindef.AbstractPlugin -

ProductionSNPCallerPluginV2 Parameters

i: /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018

k: /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/keyfile.barcoded.txt

e: ApeKI

db: /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/2016_2018_Kaki.db

o: /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/GBS_2016_2018_9May2018.vcf

eR: 0.01

d: 0

ko: false

do: true

kmerLength: 64

minPosQS: 0.0

batchSize: 8

mnQS: 0

size of all tags in tag table=2370966

size of all tissues in tissue table=0

size of all tags in mappingApproach table=2

size of all taxa in taxa table=141

ProductionSNPCallerPluginV2: Total batches to process: 1

size of all positions in snpPosition table=96277

[pool-1-thread-1] INFO net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2 -

Output VCF file:

/media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/GBS_2016_2018_9May2018.vcf

created for genotypes from this run.

size of all positions in snpPosition table=96277

size of all alleles in allele table=194649

Start processing batch 1

Enzyme: ApeKI

[pool-1-thread-1] INFO net.maizegenetics.analysis.gbs.v2.GBSUtils - /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/HKFWCCXY_8_fastq.fq: Quality score base:33

[ForkJoinPool.commonPool-worker-1] INFO net.maizegenetics.analysis.gbs.v2.GBSUtils - /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/H2J2MBCXY_1_fastq.fq: Quality score base:33

[pool-1-thread-1] ERROR net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2 - Good Barcodes Read: 10

[ForkJoinPool.commonPool-worker-1] ERROR net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2 - Good Barcodes Read: 16

java.lang.StringIndexOutOfBoundsException:

ERROR processing /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/HKFWCCXY_8_fastq.fq

Reading entry number 11 fails the length test.

Sequence length 63 minus barcode length 10 is less than kmerLength 64.

Re-run your files with either a shorter kmerLength value or a higher minimum quality score.

at net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2.processFastQ(ProductionSNPCallerPluginV2.java:362)

at net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2.processFastQFile(ProductionSNPCallerPluginV2.java:327)

at net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2.lambda$processData$2(ProductionSNPCallerPluginV2.java:252)

at java.util.stream.ForEachOps$ForEachOp$OfRef.accept(ForEachOps.java:184)

at java.util.ArrayList$ArrayListSpliterator.forEachRemaining(ArrayList.java:1382)

at java.util.stream.AbstractPipeline.copyInto(AbstractPipeline.java:481)

at java.util.stream.ForEachOps$ForEachTask.compute(ForEachOps.java:291)

at java.util.concurrent.CountedCompleter.exec(CountedCompleter.java:731)

at java.util.concurrent.ForkJoinTask.doExec(ForkJoinTask.java:289)

at java.util.concurrent.ForkJoinTask.doInvoke(ForkJoinTask.java:401)

at java.util.concurrent.ForkJoinTask.invoke(ForkJoinTask.java:734)

at java.util.stream.ForEachOps$ForEachOp.evaluateParallel(ForEachOps.java:160)

at java.util.stream.ForEachOps$ForEachOp$OfRef.evaluateParallel(ForEachOps.java:174)

at java.util.stream.AbstractPipeline.evaluate(AbstractPipeline.java:233)

at java.util.stream.ReferencePipeline.forEach(ReferencePipeline.java:418)

at java.util.stream.ReferencePipeline$Head.forEach(ReferencePipeline.java:583)

at net.maizegenetics.analysis.gbs.v2.ProductionSNPCallerPluginV2.processData(ProductionSNPCallerPluginV2.java:250)

at net.maizegenetics.plugindef.AbstractPlugin.performFunction(AbstractPlugin.java:112)

at net.maizegenetics.plugindef.AbstractPlugin.dataSetReturned(AbstractPlugin.java:1837)

at net.maizegenetics.plugindef.ThreadedPluginListener.run(ThreadedPluginListener.java:29)

at java.util.concurrent.Executors$RunnableAdapter.call(Executors.java:511)

at java.util.concurrent.FutureTask.run(FutureTask.java:266)

at java.util.concurrent.ThreadPoolExecutor.runWorker(ThreadPoolExecutor.java:1149)

at java.util.concurrent.ThreadPoolExecutor$Worker.run(ThreadPoolExecutor.java:624)

at java.lang.Thread.run(Thread.java:748)

java.lang.StringIndexOutOfBoundsException:

ERROR processing /media/stephanie/External4/GBS_2016_2018_Combined/9May2018/9May2018/H2J2MBCXY_1_fastq.fq

Reading entry number 17 fails the length test.

Sequence length 65 minus barcode length 9 is less than kmerLength 64.