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alexdobin / STAR

RNA-seq aligner
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The two mates of paired-end read are marked as both mate1 or both mate2! #609

Closed shmilyfhh closed 5 years ago

shmilyfhh commented 5 years ago

Hi there,

I am trying to combine STAR mapping and RSEM quantification to analyze paired-end RNAseq data. But when I try to feed Aligned.toTranscriptome.out.bam out from STAR with ENCODE parameters to RSEM, I get error message 'The two mates of paired-end read NS500653:81:HGHL3BGX2:2:11202:3915:6108 are marked as both mate1 or both mate2!'

I also attached the read alignment information in Aligned.toTranscriptome.out.bam after name sorting: NS500653:81:HGHL3BGX2:2:11202:3915:6108 339 ENST00000546120.1 302 76M = 286 -92 ATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACCATCGAGAACGTCAAAG EEEEEEE/EEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA NH:i:3 HI:i:3 NS500653:81:HGHL3BGX2:2:11202:3915:6108 419 ENST00000546120.1 286 76M = 302 92 CAGAGGTGGGATGCAAATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACC AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEAEE/E/EE NH:i:3 HI:i:3 NS500653:81:HGHL3BGX2:2:11202:3915:6108 83 ENST00000541272.1 308 76M = 292 -92 ATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACCATCGAGAACGTCAAAG EEEEEEE/EEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA NH:i:3 HI:i:1 NS500653:81:HGHL3BGX2:2:11202:3915:6108 339 ENST00000541272.1 308 76M = 292 -92 ATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACCATCGAGAACGTCAAAG EEEEEEE/EEEEEAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAAA NH:i:3 HI:i:2 NS500653:81:HGHL3BGX2:2:11202:3915:6108 163 ENST00000541272.1 292 76M = 308 92 CAGAGGTGGGATGCAAATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACC AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEAEE/E/EE NH:i:3 HI:i:1 NS500653:81:HGHL3BGX2:2:11202:3915:6108 419 ENST00000541272.1 292 76M = 308 92 CAGAGGTGGGATGCAAATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACC AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEAEE/E/EE NH:i:3 HI:i:2 NS500653:81:HGHL3BGX2:2:11202:3915:6108 77 0 0 CAGAGGTGGGATGCAAATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACC AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEAEE/E/EE NH:i:0 HI:i:0 AS:i:75 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:2:11202:3915:6108 141 0 0 CAGAGGTGGGATGCAAATCTTCGTGAAGACACTCACTGGCAAGACCATCACCCTTGAGGTGGAGCCCAGTGACACC AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEEEEEEEEEEEEEEEEEEEAEE/E/EE NH:i:0 HI:i:0 AS:i:75 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:2:11202:3915:6108 77 0 0 CTTTGACGTTCTCGATGGTGTCACTGGGCTCCACCTCAAGGGTGATGGTCTTGCCAGTGAGTGTCTTCACGAAGAT AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEE/EEEEEEE NH:i:0 HI:i:0 AS:i:73 nM:i:1 uT:A:1 NS500653:81:HGHL3BGX2:2:11202:3915:6108 141 0 0 CTTTGACGTTCTCGATGGTGTCACTGGGCTCCACCTCAAGGGTGATGGTCTTGCCAGTGAGTGTCTTCACGAAGAT AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEE/EEEEEEE NH:i:0 HI:i:0 AS:i:73 nM:i:1 uT:A:1

Would you help me with this issue?

thanks, huihui

alexdobin commented 5 years ago

Hi Huihui,

please try the latest versions of STAR 2.6.1d or 2.7.0f, and if the problem persists, please send me the Log.out file.

Cheers Alex

shmilyfhh commented 5 years ago

Hi Alex,

I recently installed STAR, it says "### versions versionGenome 2.7.0d". I believe this is the newest version?

STAR version=2.7.0f_0328 STAR compilation time,server,dir=Thu Mar 28 18:46:03 EDT 2019 vega:/home/dobin/data/STAR/STARcode/STAR.master/source

DEFAULT parameters:

versionGenome 2.7.0d parametersFiles -
sysShell - runMode alignReads runThreadN 1 runDirPerm User_RWX runRNGseed 777 genomeDir ./GenomeDir/ genomeLoad NoSharedMemory genomeFastaFiles -
genomeChainFiles -
genomeSAindexNbases 14 genomeChrBinNbits 18 genomeSAsparseD 1 genomeSuffixLengthMax 18446744073709551615 genomeFileSizes 0
genomeConsensusFile - readFilesType Fastx
readFilesIn Read1 Read2
readFilesPrefix - readFilesCommand -
readMatesLengthsIn NotEqual readMapNumber 18446744073709551615 readNameSeparator /
inputBAMfile - bamRemoveDuplicatesType - bamRemoveDuplicatesMate2basesN 0 limitGenomeGenerateRAM 31000000000 limitIObufferSize 150000000 limitOutSAMoneReadBytes 100000 limitOutSJcollapsed 1000000 limitOutSJoneRead 1000 limitBAMsortRAM 0 limitSjdbInsertNsj 1000000 limitNreadsSoft 18446744073709551615 outFileNamePrefix ./ outTmpDir - outTmpKeep None outStd Log outReadsUnmapped None outQSconversionAdd 0 outMultimapperOrder Old_2.4 outSAMtype SAM
outSAMmode Full outSAMstrandField None outSAMattributes Standard
outSAMunmapped None
outSAMorder Paired outSAMprimaryFlag OneBestScore outSAMreadID Standard outSAMmapqUnique 255 outSAMflagOR 0 outSAMflagAND 65535 outSAMattrRGline -
outSAMheaderHD -
outSAMheaderPG -
outSAMheaderCommentFile - outBAMcompression 1 outBAMsortingThreadN 0 outBAMsortingBinsN 50 outSAMfilter None
outSAMmultNmax 18446744073709551615 outSAMattrIHstart 1 outSAMtlen 1 outSJfilterReads All outSJfilterCountUniqueMin 3 1 1 1
outSJfilterCountTotalMin 3 1 1 1
outSJfilterOverhangMin 30 12 12 12
outSJfilterDistToOtherSJmin 10 0 5 10
outSJfilterIntronMaxVsReadN 50000 100000 200000
outWigType None
outWigStrand Stranded
outWigReferencesPrefix - outWigNorm RPM
outFilterType Normal outFilterMultimapNmax 10 outFilterMultimapScoreRange 1 outFilterScoreMin 0 outFilterScoreMinOverLread 0.66 outFilterMatchNmin 0 outFilterMatchNminOverLread 0.66 outFilterMismatchNmax 10 outFilterMismatchNoverLmax 0.3 outFilterMismatchNoverReadLmax 1 outFilterIntronMotifs None outFilterIntronStrands RemoveInconsistentStrands clip5pNbases 0
clip3pNbases 0
clip3pAfterAdapterNbases 0
clip3pAdapterSeq -
clip3pAdapterMMp 0.1
winBinNbits 16 winAnchorDistNbins 9 winFlankNbins 4 winAnchorMultimapNmax 50 winReadCoverageRelativeMin 0.5 winReadCoverageBasesMin 0 scoreGap 0 scoreGapNoncan -8 scoreGapGCAG -4 scoreGapATAC -8 scoreStitchSJshift 1 scoreGenomicLengthLog2scale -0.25 scoreDelBase -2 scoreDelOpen -2 scoreInsOpen -2 scoreInsBase -2 seedSearchLmax 0 seedSearchStartLmax 50 seedSearchStartLmaxOverLread 1 seedPerReadNmax 1000 seedPerWindowNmax 50 seedNoneLociPerWindow 10 seedMultimapNmax 10000 seedSplitMin 12 alignIntronMin 21 alignIntronMax 0 alignMatesGapMax 0 alignTranscriptsPerReadNmax 10000 alignSJoverhangMin 5 alignSJDBoverhangMin 3 alignSJstitchMismatchNmax 0 -1 0 0
alignSplicedMateMapLmin 0 alignSplicedMateMapLminOverLmate 0.66 alignWindowsPerReadNmax 10000 alignTranscriptsPerWindowNmax 100 alignEndsType Local alignSoftClipAtReferenceEnds Yes alignEndsProtrude 0 ConcordantPair
alignInsertionFlush None peOverlapNbasesMin 0 peOverlapMMp 0.01 chimSegmentMin 0 chimScoreMin 0 chimScoreDropMax 20 chimScoreSeparation 10 chimScoreJunctionNonGTAG -1 chimMainSegmentMultNmax 10 chimJunctionOverhangMin 20 chimOutType Junctions
chimFilter banGenomicN
chimSegmentReadGapMax 0 chimMultimapNmax 0 chimMultimapScoreRange 1 chimNonchimScoreDropMin 20 chimOutJunctionFormat 0
sjdbFileChrStartEnd -
sjdbGTFfile - sjdbGTFchrPrefix - sjdbGTFfeatureExon exon sjdbGTFtagExonParentTranscript transcript_id sjdbGTFtagExonParentGene gene_id sjdbOverhang 100 sjdbScore 2 sjdbInsertSave Basic varVCFfile - waspOutputMode None quantMode -
quantTranscriptomeBAMcompression 1 quantTranscriptomeBan IndelSoftclipSingleend twopass1readsN 18446744073709551615 twopassMode None soloType None soloCBstart 1 soloUMIstart 17 soloCBlen 16 soloUMIlen 10 soloBarcodeReadLength 1 soloCBwhitelist - soloStrand Forward soloOutFileNames Solo.out/ genes.tsv barcodes.tsv matrix.mtx matrixSJ.mtx
soloFeatures Gene
soloUMIdedup 1MM_All

Command Line:

STAR --genomeDir /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR --readFilesIn /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz --outSAMunmapped Within --outFilterType BySJout --outSAMattributes NH HI AS NM MD --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --readFilesCommand zcat --runThreadN 28 --genomeLoad NoSharedMemory --limitBAMsortRAM 10000000000 --outSAMtype BAM SortedByCoordinate --quantMode TranscriptomeSAM --outSAMstrandField intronMotif --outSAMheaderCommentFile commentsENCODElong.txt --outSAMheaderHD @HD VN:1.4 SO:coordinate

Initial USER parameters from Command Line:
All USER parameters from Command Line:

genomeDir /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR ~RE-DEFINED readFilesIn /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz ~RE-DEFINED outSAMunmapped Within ~RE-DEFINED outFilterType BySJout ~RE-DEFINED outSAMattributes NH HI AS NM MD ~RE-DEFINED outFilterMultimapNmax 20 ~RE-DEFINED outFilterMismatchNmax 999 ~RE-DEFINED outFilterMismatchNoverReadLmax0.04 ~RE-DEFINED alignIntronMin 20 ~RE-DEFINED alignIntronMax 1000000 ~RE-DEFINED alignMatesGapMax 1000000 ~RE-DEFINED alignSJoverhangMin 8 ~RE-DEFINED alignSJDBoverhangMin 1 ~RE-DEFINED sjdbScore 1 ~RE-DEFINED readFilesCommand zcat ~RE-DEFINED runThreadN 28 ~RE-DEFINED genomeLoad NoSharedMemory ~RE-DEFINED limitBAMsortRAM 10000000000 ~RE-DEFINED outSAMtype BAM SortedByCoordinate ~RE-DEFINED quantMode TranscriptomeSAM ~RE-DEFINED outSAMstrandField intronMotif ~RE-DEFINED outSAMheaderCommentFile commentsENCODElong.txt ~RE-DEFINED outSAMheaderHD @HD VN:1.4 SO:coordinate ~RE-DEFINED

Finished reading parameters from all sources
Final user re-defined parameters-----------------:

runThreadN 28 genomeDir /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR genomeLoad NoSharedMemory readFilesIn /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz
readFilesCommand zcat
limitBAMsortRAM 10000000000 outSAMtype BAM SortedByCoordinate
outSAMstrandField intronMotif outSAMattributes NH HI AS NM MD
outSAMunmapped Within
outSAMheaderHD @HD VN:1.4 SO:coordinate
outSAMheaderCommentFile commentsENCODElong.txt outFilterType BySJout outFilterMultimapNmax 20 outFilterMismatchNmax 999 outFilterMismatchNoverReadLmax 0.04 alignIntronMin 20 alignIntronMax 1000000 alignMatesGapMax 1000000 alignSJoverhangMin 8 alignSJDBoverhangMin 1 sjdbScore 1 quantMode TranscriptomeSAM

Final effective command line:

STAR --runThreadN 28 --genomeDir /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR --genomeLoad NoSharedMemory --readFilesIn /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz --readFilesCommand zcat --limitBAMsortRAM 10000000000 --outSAMtype BAM SortedByCoordinate --outSAMstrandField intronMotif --outSAMattributes NH HI AS NM MD --outSAMunmapped Within --outSAMheaderHD @HD VN:1.4 SO:coordinate --outSAMheaderCommentFile commentsENCODElong.txt --outFilterType BySJout --outFilterMultimapNmax 20 --outFilterMismatchNmax 999 --outFilterMismatchNoverReadLmax 0.04 --alignIntronMin 20 --alignIntronMax 1000000 --alignMatesGapMax 1000000 --alignSJoverhangMin 8 --alignSJDBoverhangMin 1 --sjdbScore 1 --quantMode TranscriptomeSAM

Final parameters after user input--------------------------------:

versionGenome 2.7.0d parametersFiles -
sysShell - runMode alignReads runThreadN 28 runDirPerm User_RWX runRNGseed 777 genomeDir /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR genomeLoad NoSharedMemory genomeFastaFiles -
genomeChainFiles -
genomeSAindexNbases 14 genomeChrBinNbits 18 genomeSAsparseD 1 genomeSuffixLengthMax 18446744073709551615 genomeFileSizes 0
genomeConsensusFile - readFilesType Fastx
readFilesIn /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz
readFilesPrefix - readFilesCommand zcat
readMatesLengthsIn NotEqual readMapNumber 18446744073709551615 readNameSeparator /
inputBAMfile - bamRemoveDuplicatesType - bamRemoveDuplicatesMate2basesN 0 limitGenomeGenerateRAM 31000000000 limitIObufferSize 150000000 limitOutSAMoneReadBytes 100000 limitOutSJcollapsed 1000000 limitOutSJoneRead 1000 limitBAMsortRAM 10000000000 limitSjdbInsertNsj 1000000 limitNreadsSoft 18446744073709551615 outFileNamePrefix ./ outTmpDir - outTmpKeep None outStd Log outReadsUnmapped None outQSconversionAdd 0 outMultimapperOrder Old_2.4 outSAMtype BAM SortedByCoordinate
outSAMmode Full outSAMstrandField intronMotif outSAMattributes NH HI AS NM MD
outSAMunmapped Within
outSAMorder Paired outSAMprimaryFlag OneBestScore outSAMreadID Standard outSAMmapqUnique 255 outSAMflagOR 0 outSAMflagAND 65535 outSAMattrRGline -
outSAMheaderHD @HD VN:1.4 SO:coordinate
outSAMheaderPG -
outSAMheaderCommentFile commentsENCODElong.txt outBAMcompression 1 outBAMsortingThreadN 0 outBAMsortingBinsN 50 outSAMfilter None
outSAMmultNmax 18446744073709551615 outSAMattrIHstart 1 outSAMtlen 1 outSJfilterReads All outSJfilterCountUniqueMin 3 1 1 1
outSJfilterCountTotalMin 3 1 1 1
outSJfilterOverhangMin 30 12 12 12
outSJfilterDistToOtherSJmin 10 0 5 10
outSJfilterIntronMaxVsReadN 50000 100000 200000
outWigType None
outWigStrand Stranded
outWigReferencesPrefix - outWigNorm RPM
outFilterType BySJout outFilterMultimapNmax 20 outFilterMultimapScoreRange 1 outFilterScoreMin 0 outFilterScoreMinOverLread 0.66 outFilterMatchNmin 0 outFilterMatchNminOverLread 0.66 outFilterMismatchNmax 999 outFilterMismatchNoverLmax 0.3 outFilterMismatchNoverReadLmax 0.04 outFilterIntronMotifs None outFilterIntronStrands RemoveInconsistentStrands clip5pNbases 0
clip3pNbases 0
clip3pAfterAdapterNbases 0
clip3pAdapterSeq -
clip3pAdapterMMp 0.1
winBinNbits 16 winAnchorDistNbins 9 winFlankNbins 4 winAnchorMultimapNmax 50 winReadCoverageRelativeMin 0.5 winReadCoverageBasesMin 0 scoreGap 0 scoreGapNoncan -8 scoreGapGCAG -4 scoreGapATAC -8 scoreStitchSJshift 1 scoreGenomicLengthLog2scale -0.25 scoreDelBase -2 scoreDelOpen -2 scoreInsOpen -2 scoreInsBase -2 seedSearchLmax 0 seedSearchStartLmax 50 seedSearchStartLmaxOverLread 1 seedPerReadNmax 1000 seedPerWindowNmax 50 seedNoneLociPerWindow 10 seedMultimapNmax 10000 seedSplitMin 12 alignIntronMin 20 alignIntronMax 1000000 alignMatesGapMax 1000000 alignTranscriptsPerReadNmax 10000 alignSJoverhangMin 8 alignSJDBoverhangMin 1 alignSJstitchMismatchNmax 0 -1 0 0
alignSplicedMateMapLmin 0 alignSplicedMateMapLminOverLmate 0.66 alignWindowsPerReadNmax 10000 alignTranscriptsPerWindowNmax 100 alignEndsType Local alignSoftClipAtReferenceEnds Yes alignEndsProtrude 0 ConcordantPair
alignInsertionFlush None peOverlapNbasesMin 0 peOverlapMMp 0.01 chimSegmentMin 0 chimScoreMin 0 chimScoreDropMax 20 chimScoreSeparation 10 chimScoreJunctionNonGTAG -1 chimMainSegmentMultNmax 10 chimJunctionOverhangMin 20 chimOutType Junctions
chimFilter banGenomicN
chimSegmentReadGapMax 0 chimMultimapNmax 0 chimMultimapScoreRange 1 chimNonchimScoreDropMin 20 chimOutJunctionFormat 0
sjdbFileChrStartEnd -
sjdbGTFfile - sjdbGTFchrPrefix - sjdbGTFfeatureExon exon sjdbGTFtagExonParentTranscript transcript_id sjdbGTFtagExonParentGene gene_id sjdbOverhang 100 sjdbScore 1 sjdbInsertSave Basic varVCFfile - waspOutputMode None quantMode TranscriptomeSAM
quantTranscriptomeBAMcompression 1 quantTranscriptomeBan IndelSoftclipSingleend twopass1readsN 18446744073709551615 twopassMode None soloType None soloCBstart 1 soloUMIstart 17 soloCBlen 16 soloUMIlen 10 soloBarcodeReadLength 1 soloCBwhitelist - soloStrand Forward soloOutFileNames Solo.out/ genes.tsv barcodes.tsv matrix.mtx matrixSJ.mtx
soloFeatures Gene
soloUMIdedup 1MM_All

Input read files for mate 1, from input string /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz -rw-r--r-- 1 huihui.fan domain users 2821900106 Apr 15 2018 /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz

readsCommandsFile: exec > "./_STARtmp/tmp.fifo.read1" echo FILE 0 zcat "/home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz"

Input read files for mate 2, from input string /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz -rw-r--r-- 1 huihui.fan domain users 2842632002 Apr 15 2018 /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz

readsCommandsFile: exec > "./_STARtmp/tmp.fifo.read2" echo FILE 0 zcat "/home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz"

WARNING --outSAMstrandField=intronMotif, therefore STAR will output XS attribute Finished loading and checking parameters Reading genome generation parameters:

STAR --runMode genomeGenerate --runThreadN 28 --genomeDir /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR --genomeFastaFiles /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/encodeDCCref/GRCh37.p13.genome.fa /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/encodeDCCref/Spikes.fixed.fasta --outFileNamePrefix /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/STAR --sjdbGTFfile /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/encodeDCCref/gencode.v19.annotation.gtf --sjdbOverhang 100

GstrandBit=32

versionGenome 2.7.0d ~RE-DEFINED genomeFastaFiles /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/encodeDCCref/GRCh37.p13.genome.fa /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/encodeDCCref/Spikes.fixed.fasta ~RE-DEFINED genomeSAindexNbases 14 ~RE-DEFINED genomeChrBinNbits 18 ~RE-DEFINED genomeSAsparseD 1 ~RE-DEFINED sjdbOverhang 100 ~RE-DEFINED sjdbFileChrStartEnd - ~RE-DEFINED sjdbGTFfile /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/encodeDCCref/gencode.v19.annotation.gtf ~RE-DEFINED sjdbGTFchrPrefix - ~RE-DEFINED sjdbGTFfeatureExon exon ~RE-DEFINED sjdbGTFtagExonParentTranscripttranscript_id ~RE-DEFINED sjdbGTFtagExonParentGene gene_id ~RE-DEFINED sjdbInsertSave Basic ~RE-DEFINED genomeFileSizes 3372012836 25250695746 ~RE-DEFINED Genome version is compatible with current STAR Number of real (reference) chromosomes= 393 1 chr1 249250621 0 2 chr2 243199373 249298944 3 chr3 198022430 492568576 4 chr4 191154276 690749440 5 chr5 180915260 882114560 6 chr6 171115067 1063256064 7 chr7 159138663 1234436096 8 chr8 146364022 1393819648 9 chr9 141213431 1540358144 10 chr10 135534747 1681653760 11 chr11 135006516 1817444352 12 chr12 133851895 1952710656 13 chr13 115169878 2086666240 14 chr14 107349540 2202009600 15 chr15 102531392 2309488640 16 chr16 90354753 2412249088 17 chr17 81195210 2502688768 18 chr18 78077248 2583953408 19 chr19 59128983 2662072320 20 chr20 63025520 2721316864 21 chr21 48129895 2784493568 22 chr22 51304566 2832728064 23 chrX 155270560 2884108288 24 chrY 59373566 3039559680 25 chrM 16569 3099066368 26 GL877870.2 66021 3099328512 27 GL877872.1 297485 3099590656 28 GL383535.1 429806 3100114944 29 JH159133.1 266316 3100639232 30 KB663606.1 305900 3101163520 31 KB021647.1 1058686 3101687808 32 KE332497.1 543325 3102998528 33 JH159131.1 393769 3103784960 34 GL949745.1 372609 3104309248 35 JH720447.1 454385 3104833536 36 GL582968.1 356330 3105357824 37 JH720446.1 97345 3105882112 38 JH591181.2 2281126 3106144256 39 JH720443.2 408430 3108503552 40 JH159134.2 3821770 3109027840 41 JH636052.4 7283150 3112960000 42 JH636053.3 1676126 3120300032 43 JH636054.1 758378 3122135040 44 JH636056.1 262912 3122921472 45 JH636058.1 716227 3123445760 46 JH636057.1 200195 3124232192 47 KE332505.1 579598 3124494336 48 JH720451.1 898979 3125280768 49 JH720452.1 522319 3126329344 50 JH720453.1 1461188 3126853632 51 JH720454.3 752267 3128426496 52 JH159136.1 200998 3129212928 53 JH806587.1 4110759 3129475072 54 JH806588.1 862483 3133669376 55 JH806589.1 270630 3134717952 56 JH806590.2 2418393 3135242240 57 JH806591.1 882083 3137863680 58 JH806592.1 835911 3138912256 59 JH806593.1 389631 3139960832 60 JH806594.1 390496 3140485120 61 JH806595.1 444074 3141009408 62 JH806596.1 413927 3141533696 63 JH806597.1 1045622 3142057984 64 JH720448.1 70483 3143106560 65 JH806598.1 899320 3143368704 66 JH806599.1 1214327 3144417280 67 JH806600.2 6530008 3145728000 68 JH806601.1 1389764 3152281600 69 JH806602.1 713266 3153854464 70 JH806573.1 24680 3154640896 71 JH806574.2 22982 3154903040 72 JH806575.1 47409 3155165184 73 JH806580.1 93149 3155427328 74 JH806583.1 167183 3155689472 75 JH806584.1 70876 3155951616 76 JH806585.1 73505 3156213760 77 JH806603.1 182949 3156475904 78 JH159150.3 3110903 3156738048 79 GL582969.1 251823 3159883776 80 JH806577.1 22394 3160145920 81 JH806578.1 169437 3160408064 82 JH806579.1 211307 3160670208 83 JH159137.1 191409 3160932352 84 KE332502.1 341712 3161194496 85 KB021645.1 1523386 3161718784 86 KB663607.2 334922 3163291648 87 KE332500.1 228602 3163815936 88 KE332496.1 503215 3164078080 89 GL383558.1 457041 3164602368 90 GL582976.1 412535 3165126656 91 GL383523.1 171362 3165650944 92 KE332498.1 149443 3165913088 93 GL949743.1 608579 3166175232 94 KE332506.1 307252 3166961664 95 GL383536.1 203777 3167485952 96 JH591184.1 462282 3167748096 97 JH636061.1 186059 3168272384 98 JH806576.1 273386 3168534528 99 GL383524.1 78793 3169058816 100 GL582973.1 321004 3169320960 101 JH159138.1 108875 3169845248 102 KB021648.1 469972 3170107392 103 JH159139.1 120441 3170631680 104 JH159140.1 546435 3170893824 105 JH591182.1 196262 3171680256 106 JH159132.1 100694 3171942400 107 JH720449.1 212298 3172204544 108 JH591183.1 177920 3172466688 109 JH720444.2 273128 3172728832 110 JH159141.2 240775 3173253120 111 KB663604.1 478993 3173515264 112 JH720455.1 65034 3174039552 113 KB021646.2 211416 3174301696 114 JH159142.2 326647 3174563840 115 JH159143.1 191402 3175088128 116 JH806582.2 342635 3175350272 117 JH159135.2 102251 3175874560 118 KE332499.1 274521 3176136704 119 GL877877.2 284527 3176660992 120 JH806586.1 43543 3177185280 121 GL582979.2 179899 3177447424 122 KB663605.1 155926 3177709568 123 GL582975.1 34662 3177971712 124 GL949744.1 276448 3178233856 125 GL383543.1 392792 3178758144 126 GL877871.1 389939 3179282432 127 GL582967.1 248177 3179806720 128 JH159149.1 245473 3180068864 129 GL582977.2 580393 3180331008 130 GL582970.1 354970 3181117440 131 GL383559.2 338640 3181641728 132 JH159144.1 388340 3182166016 133 JH591186.1 376223 3182690304 134 GL383560.1 534288 3183214592 135 GL339450.1 330164 3184001024 136 GL582971.1 1284284 3184525312 137 GL582974.1 163298 3185836032 138 JH806581.1 872115 3186098176 139 JH636060.1 437946 3187146752 140 JH591185.1 167437 3187671040 141 JH159145.1 194862 3187933184 142 GL877873.1 168465 3188195328 143 KB663608.1 283551 3188457472 144 GL582972.1 327774 3188981760 145 KB663603.1 599580 3189506048 146 KE332495.1 263861 3190292480 147 JH636059.1 295379 3190816768 148 JH720445.1 170033 3191341056 149 KE332501.1 1020827 3191603200 150 GL383561.2 644425 3192651776 151 GL949741.1 151551 3193438208 152 GL383562.1 45551 3193700352 153 GL383525.1 65063 3193962496 154 GL383544.1 128378 3194224640 155 GL383548.1 165247 3194486784 156 GL383537.1 62435 3194748928 157 GL383538.1 49281 3195011072 158 GL383516.1 49316 3195273216 159 GL383517.1 49352 3195535360 160 GL383545.1 179254 3195797504 161 GL383546.1 309802 3196059648 162 GL383547.1 154407 3196583936 163 GL877875.1 167313 3196846080 164 GL383549.1 120804 3197108224 165 GL383550.1 169178 3197370368 166 GL383551.1 184319 3197632512 167 GL877876.1 408271 3197894656 168 GL383552.1 138655 3198418944 169 GL383553.2 152874 3198681088 170 GL383554.1 296527 3198943232 171 GL383555.1 388773 3199467520 172 GL383556.1 192462 3199991808 173 GL383557.1 89672 3200253952 174 GL000258.1 1680828 3200516096 175 GL383563.2 270261 3202351104 176 GL383564.1 133151 3202875392 177 GL383565.1 223995 3203137536 178 GL383566.1 90219 3203399680 179 JH159146.1 278131 3203661824 180 JH159147.1 70345 3204186112 181 JH159148.1 88070 3204448256 182 GL383567.1 289831 3204710400 183 GL383568.1 104552 3205234688 184 GL383569.1 167950 3205496832 185 GL383570.1 164789 3205758976 186 GL383571.1 198278 3206021120 187 GL383572.1 159547 3206283264 188 GL949746.1 987716 3206545408 189 GL949747.1 729519 3207593984 190 GL949748.1 1064303 3208380416 191 GL949749.1 1091840 3209691136 192 GL949750.1 1066389 3211001856 193 GL949751.1 1002682 3212312576 194 GL949752.1 987100 3213361152 195 GL949753.1 796478 3214409728 196 GL383573.1 385657 3215458304 197 GL383574.1 155864 3215982592 198 GL383575.2 170222 3216244736 199 GL383576.1 188024 3216506880 200 GL383518.1 182439 3216769024 201 GL383519.1 110268 3217031168 202 GL383520.1 366579 3217293312 203 GL383577.1 128385 3217817600 204 GL383578.1 63917 3218079744 205 GL383579.1 201198 3218341888 206 GL383580.1 74652 3218604032 207 GL383581.1 116690 3218866176 208 GL383582.2 162811 3219128320 209 GL383583.1 96924 3219390464 210 KB663609.1 74013 3219652608 211 GL383521.1 143390 3219914752 212 GL383522.1 123821 3220176896 213 GL582966.2 96131 3220439040 214 JH636055.1 173151 3220701184 215 GL383526.1 180671 3220963328 216 GL000257.1 590426 3221225472 217 GL383527.1 164536 3222011904 218 GL383528.1 376187 3222274048 219 GL383529.1 121345 3222798336 220 GL339449.2 1612928 3223060480 221 GL383530.1 101241 3224895488 222 GL383531.1 173459 3225157632 223 GL383532.1 82728 3225419776 224 GL949742.1 226852 3225681920 225 GL383533.1 124736 3225944064 226 KB021644.1 187824 3226206208 227 GL000250.1 4622290 3226468352 228 GL000251.1 4795371 3231186944 229 GL000252.1 4610396 3236167680 230 GL000253.1 4683263 3240886272 231 GL000254.1 4833398 3245604864 232 GL000255.1 4611984 3250585600 233 GL000256.1 4928567 3255304192 234 GL383534.2 119183 3260284928 235 GL383539.1 162988 3260547072 236 GL383540.1 71551 3260809216 237 GL383541.1 171286 3261071360 238 GL383542.1 60032 3261333504 239 GL000191.1 106433 3261595648 240 GL000192.1 547496 3261857792 241 GL000193.1 189789 3262644224 242 GL000194.1 191469 3262906368 243 GL000195.1 182896 3263168512 244 GL000196.1 38914 3263430656 245 GL000197.1 37175 3263692800 246 GL000198.1 90085 3263954944 247 GL000199.1 169874 3264217088 248 GL000200.1 187035 3264479232 249 GL000201.1 36148 3264741376 250 GL000202.1 40103 3265003520 251 GL000203.1 37498 3265265664 252 GL000204.1 81310 3265527808 253 GL000205.1 174588 3265789952 254 GL000206.1 41001 3266052096 255 GL000207.1 4262 3266314240 256 GL000208.1 92689 3266576384 257 GL000209.1 159169 3266838528 258 GL000210.1 27682 3267100672 259 GL000211.1 166566 3267362816 260 GL000212.1 186858 3267624960 261 GL000213.1 164239 3267887104 262 GL000214.1 137718 3268149248 263 GL000215.1 172545 3268411392 264 GL000216.1 172294 3268673536 265 GL000217.1 172149 3268935680 266 GL000218.1 161147 3269197824 267 GL000219.1 179198 3269459968 268 GL000220.1 161802 3269722112 269 GL000221.1 155397 3269984256 270 GL000222.1 186861 3270246400 271 GL000223.1 180455 3270508544 272 GL000224.1 179693 3270770688 273 GL000225.1 211173 3271032832 274 GL000226.1 15008 3271294976 275 GL000227.1 128374 3271557120 276 GL000228.1 129120 3271819264 277 GL000229.1 19913 3272081408 278 GL000230.1 43691 3272343552 279 GL000231.1 27386 3272605696 280 GL000232.1 40652 3272867840 281 GL000233.1 45941 3273129984 282 GL000234.1 40531 3273392128 283 GL000235.1 34474 3273654272 284 GL000236.1 41934 3273916416 285 GL000237.1 45867 3274178560 286 GL000238.1 39939 3274440704 287 GL000239.1 33824 3274702848 288 GL000240.1 41933 3274964992 289 GL000241.1 42152 3275227136 290 GL000242.1 43523 3275489280 291 GL000243.1 43341 3275751424 292 GL000244.1 39929 3276013568 293 GL000245.1 36651 3276275712 294 GL000246.1 38154 3276537856 295 GL000247.1 36422 3276800000 296 GL000248.1 39786 3277062144 297 GL000249.1 38502 3277324288 298 ERCC-00002 1061 3277586432 299 ERCC-00003 1023 3277848576 300 ERCC-00004 523 3278110720 301 ERCC-00007 1135 3278372864 302 ERCC-00009 984 3278635008 303 ERCC-00012 994 3278897152 304 ERCC-00013 808 3279159296 305 ERCC-00014 1957 3279421440 306 ERCC-00016 844 3279683584 307 ERCC-00017 1136 3279945728 308 ERCC-00018 1032 3280207872 309 ERCC-00019 644 3280470016 310 ERCC-00022 751 3280732160 311 ERCC-00023 273 3280994304 312 ERCC-00024 536 3281256448 313 ERCC-00025 1994 3281518592 314 ERCC-00028 1130 3281780736 315 ERCC-00031 1138 3282042880 316 ERCC-00033 2022 3282305024 317 ERCC-00034 1019 3282567168 318 ERCC-00035 1130 3282829312 319 ERCC-00039 740 3283091456 320 ERCC-00040 744 3283353600 321 ERCC-00041 1122 3283615744 322 ERCC-00042 1023 3283877888 323 ERCC-00043 1023 3284140032 324 ERCC-00044 1156 3284402176 325 ERCC-00046 522 3284664320 326 ERCC-00048 992 3284926464 327 ERCC-00051 274 3285188608 328 ERCC-00053 1023 3285450752 329 ERCC-00054 274 3285712896 330 ERCC-00057 1021 3285975040 331 ERCC-00058 1136 3286237184 332 ERCC-00059 525 3286499328 333 ERCC-00060 523 3286761472 334 ERCC-00061 1136 3287023616 335 ERCC-00062 1023 3287285760 336 ERCC-00067 644 3287547904 337 ERCC-00069 1137 3287810048 338 ERCC-00071 642 3288072192 339 ERCC-00073 603 3288334336 340 ERCC-00074 522 3288596480 341 ERCC-00075 1023 3288858624 342 ERCC-00076 642 3289120768 343 ERCC-00077 273 3289382912 344 ERCC-00078 993 3289645056 345 ERCC-00079 644 3289907200 346 ERCC-00081 534 3290169344 347 ERCC-00083 1022 3290431488 348 ERCC-00084 994 3290693632 349 ERCC-00085 844 3290955776 350 ERCC-00086 1020 3291217920 351 ERCC-00092 1124 3291480064 352 ERCC-00095 521 3291742208 353 ERCC-00096 1107 3292004352 354 ERCC-00097 523 3292266496 355 ERCC-00098 1143 3292528640 356 ERCC-00099 1350 3292790784 357 ERCC-00104 2022 3293052928 358 ERCC-00108 1022 3293315072 359 ERCC-00109 536 3293577216 360 ERCC-00111 994 3293839360 361 ERCC-00112 1136 3294101504 362 ERCC-00113 840 3294363648 363 ERCC-00116 1991 3294625792 364 ERCC-00117 1136 3294887936 365 ERCC-00120 536 3295150080 366 ERCC-00123 1022 3295412224 367 ERCC-00126 1118 3295674368 368 ERCC-00128 1133 3295936512 369 ERCC-00130 1059 3296198656 370 ERCC-00131 771 3296460800 371 ERCC-00134 274 3296722944 372 ERCC-00136 1033 3296985088 373 ERCC-00137 537 3297247232 374 ERCC-00138 1024 3297509376 375 ERCC-00142 493 3297771520 376 ERCC-00143 784 3298033664 377 ERCC-00144 538 3298295808 378 ERCC-00145 1042 3298557952 379 ERCC-00147 1023 3298820096 380 ERCC-00148 494 3299082240 381 ERCC-00150 743 3299344384 382 ERCC-00154 537 3299606528 383 ERCC-00156 494 3299868672 384 ERCC-00157 1019 3300130816 385 ERCC-00158 1027 3300392960 386 ERCC-00160 743 3300655104 387 ERCC-00162 523 3300917248 388 ERCC-00163 543 3301179392 389 ERCC-00164 1022 3301441536 390 ERCC-00165 872 3301703680 391 ERCC-00168 1024 3301965824 392 ERCC-00170 1023 3302227968 393 ERCC-00171 505 3302490112 --sjdbOverhang = 100 taken from the generated genome Started loading the genome: Thu Apr 4 19:52:24 2019

Genome: size given as a parameter = 3372012836 SA: size given as a parameter = 25250695746 SAindex: size given as a parameter = 1 Read from SAindex: pGe.gSAindexNbases=14 nSAi=357913940 nGenome=3372012836; nSAbyte=25250695746 GstrandBit=32 SA number of indices=6121380786 Shared memory is not used for genomes. Allocated a private copy of the genome. Genome file size: 3372012836 bytes; state: good=1 eof=0 fail=0 bad=0 Loading Genome ... done! state: good=1 eof=0 fail=0 bad=0; loaded 3372012836 bytes SA file size: 25250695746 bytes; state: good=1 eof=0 fail=0 bad=0 Loading SA ... done! state: good=1 eof=0 fail=0 bad=0; loaded 25250695746 bytes Loading SAindex ... done: 1565873619 bytes Finished loading the genome: Thu Apr 4 19:52:38 2019

Processing splice junctions database sjdbN=344580, pGe.sjdbOverhang=100 To accommodate alignIntronMax=1000000 redefined winBinNbits=18 To accommodate alignIntronMax=1000000 and alignMatesGapMax=1000000, redefined winFlankNbins=4 and winAnchorDistNbins=8 Loaded transcript database, nTr=196520 Loaded exon database, nEx=1196293 Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread0 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread0 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread1 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread1 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread2 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread2 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread3 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread3 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread4 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread4 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread5 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread5 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread6 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread6 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread7 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread7 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread8 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread8 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread9 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread9 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread10 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread10 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread11 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread11 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread12 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread12 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread13 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread13 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread14 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread14 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread15 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread15 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread16 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread16 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread17 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread17 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread18 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread18 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread19 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread19 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread20 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread20 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread21 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread21 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread22 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread22 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread23 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread23 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread24 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread24 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread25 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread25 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread26 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread26 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate1.thread27 ... ok Opening the file: ./_STARtmp//FilterBySJoutFiles.mate2.thread27 ... ok Created thread # 1 Created thread # 2 Created thread # 3 Created thread # 4 Starting to map file # 0 mate 1: /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r1.fq.gz mate 2: /home/huihui.fan/shen-secondary/projects/2016_05_19_Jitu_LMS/RNAseq/fastq/MP120N.r2.fq.gz Created thread # 5 Created thread # 6 Created thread # 7 Created thread # 8 Created thread # 9 Created thread # 10 Created thread # 11 Created thread # 12 Created thread # 13 Created thread # 14 Created thread # 15 Created thread # 16 Created thread # 17 Created thread # 18 Created thread # 19 Created thread # 20 Created thread # 21 Created thread # 22 Created thread # 23 Created thread # 24 Created thread # 25 Created thread # 26 Created thread # 27 BAM sorting: 263703 mapped reads BAM sorting bins genomic start loci: 1 0 17222535 2 0 91411055 3 0 169199971 4 1 32189385 5 1 122098131 6 1 238267689 7 2 62281422 8 2 171759107 9 3 85706516 10 4 14430010 11 4 122881433 12 5 34284456 13 5 52860534 14 5 56334700 15 5 152382184 16 6 80458455 17 7 20122704 18 8 9442120 19 8 112564469 20 9 48944491 21 9 122114177 22 10 65158051 23 10 117075376 24 11 54675230 25 12 33919005 26 13 35015939 27 13 50053325 28 13 50053354 29 13 50053397 30 13 50053449 31 13 50282761 32 13 50329290 33 13 50329365 34 13 50329419 35 13 50329446 36 13 50329484 37 14 30012653 38 14 96818868 39 15 57100633 40 16 18967236 41 16 19091465 42 16 42284076 43 18 3449118 44 19 2658082 45 21 22675471 46 22 70906517 47 24 6917 48 41 629272 Thread #20 end of input stream, nextChar=-1 Completed: thread #15 Completed: thread #27 Completed: thread #25 Completed: thread #13 Completed: thread #11 Completed: thread #10 Completed: thread #17 Completed: thread #14 Completed: thread #16 Completed: thread #12 Completed: thread #23 Completed: thread #22 Completed: thread #26 Completed: thread #21 Completed: thread #0 Completed: thread #24 Completed: thread #3 Completed: thread #4 Completed: thread #2 Completed: thread #5 Completed: thread #1 Joined thread # 1 Joined thread # 2 Joined thread # 3 Joined thread # 4 Joined thread # 5 Completed: thread #20 Completed: thread #6 Joined thread # 6 Completed: thread #9 Completed: thread #18 Completed: thread #7 Joined thread # 7 Completed: thread #19 Completed: thread #8 Joined thread # 8 Joined thread # 9 Joined thread # 10 Joined thread # 11 Joined thread # 12 Joined thread # 13 Joined thread # 14 Joined thread # 15 Joined thread # 16 Joined thread # 17 Joined thread # 18 Joined thread # 19 Joined thread # 20 Joined thread # 21 Joined thread # 22 Joined thread # 23 Joined thread # 24 Joined thread # 25 Joined thread # 26 Joined thread # 27 Completed stage 1 mapping of outFilterBySJout mapping Detected 87014 novel junctions that passed filtering, will proceed to filter reads that contained unannotated junctions Created thread # 1 Created thread # 2 Created thread # 3 Created thread # 4 Created thread # 5 Created thread # 6 Created thread # 7 Created thread # 8 Created thread # 9 Created thread # 10 Created thread # 11 Created thread # 12 Created thread # 13 Created thread # 14 Created thread # 15 Created thread # 16 Created thread # 17 Created thread # 18 Created thread # 19 Created thread # 20 Created thread # 21 Created thread # 22 Created thread # 23 Created thread # 24 Created thread # 25 Created thread # 26 Created thread # 27 Completed: thread #15 Completed: thread #20 Completed: thread #13 Completed: thread #18 Completed: thread #16 Completed: thread #17 Completed: thread #8 Completed: thread #12 Completed: thread #26 Completed: thread #3 Completed: thread #11 Completed: thread #21 Completed: thread #19 Completed: thread #23 Completed: thread #4 Completed: thread #14 Completed: thread #22 Completed: thread #10 Completed: thread #7 Completed: thread #1 Completed: thread #2 Completed: thread #5 Completed: thread #9 Completed: thread #25 Completed: thread #6 Completed: thread #0 Joined thread # 1 Joined thread # 2 Joined thread # 3 Joined thread # 4 Joined thread # 5 Joined thread # 6 Joined thread # 7 Joined thread # 8 Joined thread # 9 Joined thread # 10 Joined thread # 11 Joined thread # 12 Joined thread # 13 Joined thread # 14 Joined thread # 15 Joined thread # 16 Joined thread # 17 Joined thread # 18 Joined thread # 19 Joined thread # 20 Joined thread # 21 Joined thread # 22 Joined thread # 23 Completed: thread #24 Joined thread # 24 Joined thread # 25 Joined thread # 26 Completed: thread #27 Joined thread # 27 Apr 04 20:03:32 ..... started sorting BAM Max memory needed for sorting = 1090764200 ALL DONE!

Thanks, huihui

JohnMCMa commented 5 years ago

When I attempted to diagnose the same issue on my end, I noticed it may be a problem on RSEM's side (convert-sam-for-rsem to be exact). Have you processed the .Aligned.toTranscriptome.out.bam through convert-sam-for-rsem before using it for rsem-calculate-expression?

shmilyfhh commented 5 years ago

Yes, I did.

From: John Ma notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Tuesday, May 28, 2019 at 4:48 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Author author@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

When I attempted to diagnose the same issue on my end, I noticed it may be a problem on RSEM's side (convert-sam-for-rsem to be exact). Have you processed the .Aligned.toTranscriptome.out.bam through convert-sam-for-rsem before using it for rsem-calculate-expression?

— You are receiving this because you authored the thread. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/609?email_source=notifications&email_token=AEFKUAUW7MICB3DI2K7FDYLPXWLCBA5CNFSM4HD5HCJ2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGODWNMVKQ#issuecomment-496683690, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AEFKUAV2XAGAXY6CYIGM3Y3PXWLCBANCNFSM4HD5HCJQ.

JohnMCMa commented 5 years ago

@shmilyfhh , can you try whether not using convert-sam-for-rsem would resolve the issue? It seems like to be the case for me.

shmilyfhh commented 5 years ago

If I don’t convert before I call abundance, it will not take the file from STAR sometimes. And with this convert function, sometimes it fails with the warning I posted. So my guess would be something in the bam file from STAR is unexpected.

From: John Ma notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Tuesday, May 28, 2019 at 5:22 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

@shmilyfhhhttps://github.com/shmilyfhh , can you try whether not using convert-sam-for-rsem would resolve the issue? It seems like to be the case for me.

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alexdobin commented 5 years ago

Hi @shmilyfhh

if you do not do the conversion and feed the Aligned.toTranscriptome.out.bam directly to RSEM, how does it complain?

Cheers Alex

shmilyfhh commented 5 years ago

rsem-parse-alignments /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/RSEM/RSEMref Quant.temp/Quant Quant.stat/Quant Aligned.toTranscriptome.out.bam 3 -tag XM Read NS500653:81:HGHL3BGX2:1:11202:17968:8360 is both unalignable and alignable according to the input file! "rsem-parse-alignments /home/huihui.fan/shen-secondary/projects/tmp/genomes/hg19/RSEM/RSEMref Quant.temp/Quant Quant.stat/Quant Aligned.toTranscriptome.out.bam 3 -tag XM" failed! Plase check if you provide correct parameters/options for the pipeline!

This is the what I got when I feed Aligned.toTranscriptome.out.bam directly to RSEM.

Thanks, Huihui

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Tuesday, May 28, 2019 at 6:12 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi @shmilyfhhhttps://github.com/shmilyfhh

if you do not do the conversion and feed the Aligned.toTranscriptome.out.bam directly to RSEM, how does it complain?

Cheers Alex

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alexdobin commented 5 years ago

Hi Huihui

you are running it with the same parameters as before? With STAR version 2.7.0f? Have you trimmed the reads before mapping? If possible, please send me the Aligned.toTranscriptome.out.bam file, or grep the offending read from it (NS500653:81:HGHL3BGX2:1:11202:17968:8360)

Thanks! Alex

shmilyfhh commented 5 years ago

Hi Alex,

Thanks for getting back to me. Attached please find the read information. I did not do the trim before mapping though.

Huihui

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Wednesday, May 29, 2019 at 9:58 AM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi Huihui

you are running it with the same parameters as before? With STAR version 2.7.0f? Have you trimmed the reads before mapping? If possible, please send me the Aligned.toTranscriptome.out.bam file, or grep the offending read from it (NS500653:81:HGHL3BGX2:1:11202:17968:8360)

Thanks! Alex

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/609?email_source=notifications&email_token=AEFKUAXSXSSWEELIL5VCVVTPX2DZDA5CNFSM4HD5HCJ2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGODWPNIUA#issuecomment-496948304, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AEFKUAUAC6UTVKJ6I2PU6M3PX2DZDANCNFSM4HD5HCJQ.

shmilyfhh commented 5 years ago

I am using the same parameters with STAR version 2.7.0f.

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Wednesday, May 29, 2019 at 9:58 AM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi Huihui

you are running it with the same parameters as before? With STAR version 2.7.0f? Have you trimmed the reads before mapping? If possible, please send me the Aligned.toTranscriptome.out.bam file, or grep the offending read from it (NS500653:81:HGHL3BGX2:1:11202:17968:8360)

Thanks! Alex

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/609?email_source=notifications&email_token=AEFKUAXSXSSWEELIL5VCVVTPX2DZDA5CNFSM4HD5HCJ2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGODWPNIUA#issuecomment-496948304, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AEFKUAUAC6UTVKJ6I2PU6M3PX2DZDANCNFSM4HD5HCJQ.

alexdobin commented 5 years ago

Hi Huihui,

If possible, please send me the Aligned.toTranscriptome.out.bam file, or grep the offending read from it (NS500653:81:HGHL3BGX2:1:11202:17968:8360) .

Cheers Alex

shmilyfhh commented 5 years ago

Hi Alex,

Attached please find the read information. I am pasting the information here as well just in case you did not receive the attachment; NS500653:81:HGHL3BGX2:1:11202:17968:8360 77 0 0 0 0 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// NH:i:0 HI:i:0 AS:i:74 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 141 0 0 0 0 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// NH:i:0 HI:i:0 AS:i:74 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 77 0 0 0 0 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// NH:i:0 HI:i:0 AS:i:74 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 141 0 0 0 0 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// NH:i:0 HI:i:0 AS:i:74 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 77 0 0 0 0 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// NH:i:0 HI:i:0 AS:i:74 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 141 0 0 0 0 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// NH:i:0 HI:i:0 AS:i:74 nM:i:0 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 163 ENST00000602319.1 1961 255 76M = 2197 312 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:1 HI:i:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 83 ENST00000602319.1 2197 255 76M = 1961 -312 TTCAAGTGCCAGGAATCTACCACAAAGTAGCCGGGAGGTGCAGGGCAGCAGGGGGCACAGTGAAGTGCTGAAGGGC //EE<AAE/EEEEEEAEEEEEEEEAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEE6EE6EEAAAAA NH:i:1 HI:i:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 77 0 0 0 0 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:0 HI:i:0 AS:i:72 nM:i:1 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 141 0 0 0 0 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:0 HI:i:0 AS:i:72 nM:i:1 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 77 0 0 0 0 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:0 HI:i:0 AS:i:72 nM:i:1 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 141 0 0 0 0 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:0 HI:i:0 AS:i:72 nM:i:1 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 77 0 0 0 0 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:0 HI:i:0 AS:i:72 nM:i:1 uT:A:1 NS500653:81:HGHL3BGX2:1:11202:17968:8360 141 0 0 0 0 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ NH:i:0 HI:i:0 AS:i:72 nM:i:1 uT:A:1

Thanks, Huihui

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Monday, June 3, 2019 at 4:21 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi Huihui,

If possible, please send me the Aligned.toTranscriptome.out.bam file, or grep the offending read from it (NS500653:81:HGHL3BGX2:1:11202:17968:8360) .

Cheers Alex

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alexdobin commented 5 years ago

Hi Huihui,

something is strange with this read - it appears to have two types of entries, one correctly mapped, and multiple lines that are not mapped. Also, the sequences of the unmapped mates are exactly the same and one of them is different from the sequences of the mapped mates.

I suspect the problem is in the FASTQ files, with two different reads having the same sequence for some reason. Could you please scan your fastq files for these reads, i.e. $ zcat R1.fq.gz | grep -A3 NS500653:81:HGHL3BGX2:1:11202:17968:8360 $ zcat R2.fq.gz | grep -A3 NS500653:81:HGHL3BGX2:1:11202:17968:8360

Cheers Alex

shmilyfhh commented 5 years ago

Hi Alex,

Attached please find the read information from FASTQ files. It seems like two different read sequences have the same read name.

Thanks, Huihui

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Tuesday, June 4, 2019 at 5:35 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi Huihui,

something is strange with this read - it appears to have two types of entries, one correctly mapped, and multiple lines that are not mapped. Also, the sequences of the unmapped mates are exactly the same and one of them is different from the sequences of the mapped mates.

I suspect the problem is in the FASTQ files, with two different reads having the same sequence for some reason. Could you please scan your fastq files for these reads, i.e. $ zcat R1.fq.gz | grep -A3 NS500653:81:HGHL3BGX2:1:11202:17968:8360 $ zcat R2.fq.gz | grep -A3 NS500653:81:HGHL3BGX2:1:11202:17968:8360

Cheers Alex

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alexdobin commented 5 years ago

Hi Huihui

I cannot see the attachment, I think you need to attach it on the github website, the reply with attachments does not work...

Two reads cannot have the same name, this will screw up most tools. How did you get these fastq files? Did you pre-process them in any way?

Cheers Alex

shmilyfhh commented 5 years ago

Hi Alex,

I will paste it here instead of using attachments then. I need to double check, but I believe those data are generated by our CORE but they only provided me bam files, so I did a bam2fastq transformation to get the fastq files.

R1.fastq @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ @NS500653:81:HGHL3BGX2:1:11202:17968:8360/1 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/

R2.fastq @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 GCCCTTCAGCACTTCACTGTGCCCCCTGCTGCCCTGCACCTCCCGGCTACTTTGTGGTAGATTCCTGGCACTTGAA + AAAAAEE6EE6EEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAAAEEEEEEEEAEEEEEE/EAA<EE// @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/ @NS500653:81:HGHL3BGX2:1:11202:17968:8360/2 CTTATATCTGTTGGGATTCCCACATCTTCTAGTTGGACAATATTGGAGAAGACACCACATTTTAACTGAGTTCCAG + AAAAAEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEEAEEEEEEE/EEAEEEEEEEAEEEEEEEEEEEE/

Thanks, Huihui

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Wednesday, June 5, 2019 at 1:09 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi Huihui

I cannot see the attachment, I think you need to attach it on the github website, the reply with attachments does not work...

Two reads cannot have the same name, this will screw up most tools. How did you get these fastq files? Did you pre-process them in any way?

Cheers Alex

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/609?email_source=notifications&email_token=AEFKUAUT2FH5PWUN67LJBP3PY7XLBA5CNFSM4HD5HCJ2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGODXAMHUQ#issuecomment-499172306, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AEFKUAR5BM7JHZVUIBL5X7TPY7XLBANCNFSM4HD5HCJQ.

shmilyfhh commented 5 years ago

Hi Alex,

Sorry for all the trouble, I will get the original data and rerun the whole process. Thank you so much for your help, I appreciate it!

Thanks, Huihui

From: Alexander Dobin notifications@github.com Reply-To: alexdobin/STAR reply@reply.github.com Date: Wednesday, June 5, 2019 at 1:09 PM To: alexdobin/STAR STAR@noreply.github.com Cc: "Fan, Huihui" Huihui.Fan@vai.org, Mention mention@noreply.github.com Subject: Re: [alexdobin/STAR] The two mates of paired-end read are marked as both mate1 or both mate2! (#609)

Hi Huihui

I cannot see the attachment, I think you need to attach it on the github website, the reply with attachments does not work...

Two reads cannot have the same name, this will screw up most tools. How did you get these fastq files? Did you pre-process them in any way?

Cheers Alex

— You are receiving this because you were mentioned. Reply to this email directly, view it on GitHubhttps://github.com/alexdobin/STAR/issues/609?email_source=notifications&email_token=AEFKUAUT2FH5PWUN67LJBP3PY7XLBA5CNFSM4HD5HCJ2YY3PNVWWK3TUL52HS4DFVREXG43VMVBW63LNMVXHJKTDN5WW2ZLOORPWSZGODXAMHUQ#issuecomment-499172306, or mute the threadhttps://github.com/notifications/unsubscribe-auth/AEFKUAR5BM7JHZVUIBL5X7TPY7XLBANCNFSM4HD5HCJQ.

alexdobin commented 5 years ago

Hi Huihui,

sure, starting with the raw data is probably the best way.

Cheers Alex

lubicks commented 1 year ago

Hello - has this been resolved? I am having the same issues as described here and can't seem to solve it!

alexdobin commented 1 year ago

Please start a new issue.