I am getting this QoRTs error after cleaning the data by Trimmomatic and aligning the pair-ends reads by STAR. Please let me know where I am going wrong. I really appreciate your help. My commands:-
java -Xmx16G -jar /work/LAS/vollbrec-lab/sharmistha/QoRTs.jar QC --generatePlots --stranded --verbose --maxReadLength 90 --genomeFA /work/LAS/vollbrec-lab/sharmistha/star_files/GenomeFiles/GCA_000005005.6_B73_RefGen_v4_genomic.fna.gz --rawfastq /work/LAS/vollbrec-lab/sharmistha/NB-rep-1_ATCACG_L001_R1_001.fastq.gz,/work/LAS/vollbrec-lab/sharmistha/NB-rep-1_ATCACG_L001_R2_001.fastq.gz --generateSeparatePlots --outfilePrefix ${output} --chromSizes /work/LAS/vollbrec-lab/sharmistha/star_files/GenomeDirectory/chrLength.txt --addFunctions mismatchEngine,annotatedSpliceExonCounts,FPKM,writeGeneBodyIv,fastqUtils,referenceMatch,writeDocs,makeJunctionBed,makeAllBrowserTracks,calcDetailedGeneCounts /work/LAS/vollbrec-lab/sharmistha/star_files/star_output_files/NB-rep-1_ATCACG_L001/${input1} /work/LAS/vollbrec-lab/sharmistha/star_files/GenomeFiles/GCA_000005005.6_B73_RefGen_v4_genomic.gtf.gz /work/LAS/vollbrec-lab/sharmistha/QoRTs_files/${output}/
Starting QoRTs v1.3.0 (Compiled Fri Oct 20 11:56:37 EDT 2017) Starting time: (Mon Jul 02 14:04:41 CDT 2018) INPUT_COMMAND(QC) INPUT_ARG(infile)=/work/LAS/vollbrec-lab/sharmistha/star_files/star_output_files/NB-rep-1_ATCACG_L001/NB-rep-1_ATCACG_L001Aligned.sortedByCoord.out.bam INPUT_ARG(gtffile)=/work/LAS/vollbrec-lab/sharmistha/star_files/GenomeFiles/GCA_000005005.6_B73_RefGen_v4_genomic.gtf.gz INPUT_ARG(outdir)=/work/LAS/vollbrec-lab/sharmistha/QoRTs_files/NB-rep-1_ATCACG_L001Aligned/ INPUT_ARG(generatePlots)=true INPUT_ARG(stranded)=true INPUT_ARG(verbose)=true INPUT_ARG(maxReadLength)=Some(90) INPUT_ARG(genomeFA)=Some(List(/work/LAS/vollbrec-lab/sharmistha/star_files/GenomeFiles/GCA_000005005.6_B73_RefGen_v4_genomic.fna.gz)) INPUT_ARG(rawfastq)=Some(List(/work/LAS/vollbrec-lab/sharmistha/NB-rep-1_ATCACG_L001_R1_001.fastq.gz, /work/LAS/vollbrec-lab/sharmistha/NB-rep-1_ATCACG_L001_R2_001.fastq.gz)) INPUT_ARG(generateSeparatePlots)=true INPUT_ARG(outfilePrefix)=NB-rep-1_ATCACG_L001Aligned INPUT_ARG(chromSizes)=Some(/work/LAS/vollbrec-lab/sharmistha/star_files/GenomeDirectory/chrLength.txt) INPUT_ARG(addFunctions)=List(mismatchEngine, annotatedSpliceExonCounts, FPKM, writeGeneBodyIv, fastqUtils, referenceMatch, writeDocs, makeJunctionBed, makeAllBrowserTracks, calcDetailedGeneCounts) Created Log File: /work/LAS/vollbrec-lab/sharmistha/QoRTs_files/NB-rep-1_ATCACG_L001Aligned//NB-rep-1_ATCACG_L001AlignedQC.6JZ9kTs4cAFB.log Starting QC [Time: 2018-07-02 14:04:41] [Mem usage: [73MB / 2024MB]] [Elapsed Time: 00:00:00.0000] QoRTs is Running in paired-end mode. QoRTs is Running in any-sorted mode. Chromosome size file added. Adding target wiggle plot generation. Raw fastq files specified. Adding fastq testing. Parameter --genomeFA found. Adding reference mismatch testing. Running functions: CigarOpDistribution, FPKM, GCDistribution, GeneCalcs, InsertSize, JunctionCalcs, NVC, QualityScoreDistribution, StrandCheck, annotatedSpliceExonCounts, calcDetailedGeneCounts, chromCounts, cigarLocusCounts, fastqUtils, makeAllBrowserTracks, makeJunctionBed, makeWiggles, mismatchEngine, overlapMatch, readLengthDistro, referenceMatch, writeBiotypeCounts, writeClippedNVC, writeDESeq, writeDEXSeq, writeDocs, writeGeneBody, writeGeneBodyIv, writeGeneCounts, writeGenewiseGeneBody, Checking first 10000 reads. Checking SAM file for formatting errors... Stats on the first 10000 reads: Num Reads Primary Map: 9103 Num Reads Paired-ended: 10000 Num Reads mapped pair: 9098 Num Pair names found: 4797 Num Pairs matched: 4301 Read Seq length: 36 to 90 Unclipped Read length: 36 to 90 Final maxReadLength: 90 maxPhredScore: 41 minPhredScore: 2 NOTE: Read length is not consistent. In the first 10000 reads, read length varies from 36 to 90 (param maxReadLength=90) Note that using data that is hard-clipped prior to alignment is NOT recommended, because this makes it difficult (or impossible) to determine the sequencer read-cycle of each nucleotide base. This may obfuscate cycle-specific artifacts, trends, or errors, the detection of which is one of the primary purposes of QoRTs!In addition, hard clipping (whether before or after alignment) removes quality score data, and thus quality score metrics may be misleadingly optimistic. A MUCH preferable method of removing undesired sequence is to replace such sequence with N's, which preserves the quality score and the sequencer cycle information. Note: Data appears to be paired-ended. Sorting Note: Reads are not sorted by name (This is OK). Sorting Note: Reads are sorted by position (This is OK). Done checking first 10000 reads. No major problems detected! Starting getSRPairIterResorted... SAMRecord Reader Generated. Read length: 90. [Time: 2018-07-02 14:04:44] [Mem usage: [285MB / 2552MB]] [Elapsed Time: 00:00:03.0401] Starting fastq readthrough.
Init FastqGC Utility Init FastqQualityScore Utility Init FastqNVC Utility ============================FATAL_ERROR============================ QoRTs encountered a FATAL ERROR. For general help, use command: java -jar path/to/jar/QoRTs.jar --man ============================FATAL_ERROR============================ Error info: Exception in thread "main" java.lang.ArrayIndexOutOfBoundsException: 100 at qcUtils.fqcGC.runOnReadPair(fqcGC.scala:43) at qcUtils.runAllQC$.$anonfun$runOnSeqFile$5(runAllQC.scala:1187) at scala.collection.Iterator.foreach(Iterator.scala:929) at scala.collection.Iterator.foreach$(Iterator.scala:929) at scala.collection.AbstractIterator.foreach(Iterator.scala:1417) at scala.collection.IterableLike.foreach(IterableLike.scala:71) at scala.collection.IterableLike.foreach$(IterableLike.scala:70) at scala.collection.AbstractIterable.foreach(Iterable.scala:54) at qcUtils.runAllQC$.$anonfun$runOnSeqFile$4(runAllQC.scala:1187) at qcUtils.runAllQC$.$anonfun$runOnSeqFile$4$adapted(runAllQC.scala:1186) at scala.collection.Iterator.foreach(Iterator.scala:929) at scala.collection.Iterator.foreach$(Iterator.scala:929) at scala.collection.AbstractIterator.foreach(Iterator.scala:1417) at qcUtils.runAllQC$.runOnSeqFile(runAllQC.scala:1186) at qcUtils.runAllQC$.run(runAllQC.scala:960) at qcUtils.runAllQC$allQC_runner.run(runAllQC.scala:672) at runner.runner$.main(runner.scala:97) at runner.runner.main(runner.scala)