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hartleys / QoRTs

Quality of RNA-Seq Toolset
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long read QC #57

Open ehhill opened 6 years ago

ehhill commented 6 years ago

Hi there, I have long read RNA-seq data from the minion platform. I'm trying to plot gene body coverage of these reads. Is there a reason this data wouldn't be compatible with the QoRTs java application? When I try to run it I get this error message:

Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Record 1, Read name 1_323911, Zero-length read without FZ, CS or CQ tag

Cheers, Erin

hartleys commented 6 years ago

What sort of file do you have? It is a valid SAM file?

On Feb 22, 2018 10:58 PM, "ehhill" notifications@github.com wrote:

Hi there, I have long read RNA-seq data from the minion platform. I'm trying to plot gene body coverage of these reads. Is there a reason this data wouldn't be compatible with the QoRTs java application? When I try to run it I get this error message:

Exception in thread "main" net.sf.samtools.SAMFormatException: SAM validation error: ERROR: Record 1, Read name 1_323911, Zero-length read without FZ, CS or CQ tag

Cheers, Erin

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ehhill commented 6 years ago

I've tried SAM/BAM files generated from both gmap and minimap2 (just the generic SAM file format output by these programs). I get the same error message for both.

hartleys commented 6 years ago

Can you send the first read line of one of these files?

The SAM file parser comes from the samtools jdk, and that is what's throwing an error.

On Feb 26, 2018 5:26 PM, "ehhill" notifications@github.com wrote:

I've tried SAM/BAM files generated from both gmap and minimap2 (just the generic SAM file format output by these programs). I get the same error message for both.

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ehhill commented 6 years ago

Hi, this is the first entry from a minimap2 file.

b66c0d3f-f3e9-46d4-9556-90424e2ca7cb 4 0 0 0 0 TTGTGCTTTTCATTTATTCCTCGGCTGGGTTGTTTAGCATTCCCTGCTCTTTTTAAAACGTCATTGTTTATCCGGCTTGTCCGCAGCGGCTGGCTTTTCATCCATACCCAAACGCCTATCACCGTATGTCATCTCGTCATTTCCTGGTATCATTCTGACGTACTCGGCGTGCCATGCGGCACTTCCAGTGGCGTCATCTCCACAAGGCTCCATTTCCCTTTGCCAGGTAGGCAATCACCGTCGCCATCATCTCATACCCTCGATATGCAAAAGCATCGGGCTCGTTGCATTCGCCTCTGCCCATTCGATCATCAGGTCATGTGTACGCCCACCGGCGCTGGCTGTTCGGCATTCGACCGCTCGGCATATCTCGGCTCGACCAATCGGTGCTGGCCACATCTGCCGCCGCAATTATATGGTGCTGCGGTCTGGCCGCG $))($%-130%#2,%'/-('#$%%%%,,+..,12)'',&3)-)((#%'&(++)'++1'&'()%''%11-23(&++,9'#%$%%(((#%%%%&)''&2.,,)5,+))&'$'%&'"(&#$%&-,'))()&,/'01,1)'.(&'&$''&$+$%$&$+,,+-))'&-,'((,+).$(-+:.'.:-/+(-%+$%%('#%'')%&-/)/8+2-&+0(%0(&,)'%%'/-3(,&-&%)%)(-00%$$'#)-)()..+&&$&+0-&+-11,,/1.,).)'('/./'55,+)./-+060)&'&0-)))2()'&%$($)('$<(+/,.'''&#''))('/:/.'00%(&'$//'('(&)+',/7:;:7-(2*(&()''&%'+('',5+($%''3('))+-($&&#&)((%)(&&%&

hartleys commented 6 years ago

It looks like this is an unaligned SAM file. QoRTs only works on aligned data.

On Feb 26, 2018 8:04 PM, "ehhill" notifications@github.com wrote:

Hi, this is the first entry from a minimap2 file.

b66c0d3f-f3e9-46d4-9556-90424e2ca7cb 4 0 0 0 0 TTGTGCTTTTCATTTATTCCTCGGCTGGGTTGTTTAGCATTCCCTGCTCTTTTTAAAACG TCATTGTTTATCCGGCTTGTCCGCAGCGGCTGGCTTTTCATCCATACCCAAACGCCTATC ACCGTATGTCATCTCGTCATTTCCTGGTATCATTCTGACGTACTCGGCGTGCCATGCGGC ACTTCCAGTGGCGTCATCTCCACAAGGCTCCATTTCCCTTTGCCAGGTAGGCAATCACCG TCGCCATCATCTCATACCCTCGATATGCAAAAGCATCGGGCTCGTTGCATTCGCCTCTGC CCATTCGATCATCAGGTCATGTGTACGCCCACCGGCGCTGGCTGTTCGGCATTCGACCGC TCGGCATATCTCGGCTCGACCAATCGGTGCTGGCCACATCTGCCGCCGCAATTATATGGTGCTGCGGTCTGGCCGCG $))($%-130%#2 https://github.com/hartleys/QoRTs/issues/2 ,%'/-('#$%%%%,,+..,12)'',&3)-)((#%'&(++)'++1'&'()%''%11-23(& ++,9'#%$%%(((#%%%%&)''&2.,,)5,+))&'$'%&'"(&#$%&-,'))()&,/'01,1)'.( &'&$''&$+$%$&$+,,+-))'&-,'((,+).$(-+:.'.:-/+(-%+$%%('#%'')%&-/)/8+2-&+ 0(%0(&,)'%%'/-3(,&-&%)%)(-00%$$'#)-)()..+&&$& +0-&+-11,,/1.,).)'('/./'55,+)./-+060)&'&0-)))2()'&%$($)('$<(+/,.' ''&#''))('/:/.'00%(&'$//'('(&)+',/7:;:7-(2*(&()''&%'+('',5+($%''3( '))+-($&&#&)((%)(&&%&

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ehhill commented 6 years ago

That's odd, it should be aligned to the genome; it was from a minimap2 spliced alignment to the genome. Regardless, I've tried other sam files from gmap and these throw this error:

Error info: Exception in thread "main" java.lang.UnsupportedOperationException: empty.min at scala.collection.TraversableOnce.min(TraversableOnce.scala:222) at scala.collection.TraversableOnce.min$(TraversableOnce.scala:220) at scala.collection.mutable.ArrayOps$ofByte.min(ArrayOps.scala:203) at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3(commonSeqUtils.scala:710) at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3$adapted(commonSeqUtils.scala:709) at scala.collection.TraversableLike.$anonfun$map$1(TraversableLike.scala:234) at scala.collection.immutable.List.foreach(List.scala:389) at scala.collection.TraversableLike.map(TraversableLike.scala:234) at scala.collection.TraversableLike.map$(TraversableLike.scala:227) at scala.collection.immutable.List.map(List.scala:295) at internalUtils.commonSeqUtils$.initSamRecordIterator(commonSeqUtils.scala:709) at qcUtils.runAllQC$.runOnSeqFile(runAllQC.scala:1020) 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)

Here is an entry in this sam file. 1_377103 16 CS10_Chromosome_05 746131 40 12S5M1I7M4D54M1I7M1I5M2D9M24S 0 0 CTGATTCGCCGTGACTCTTTGCTTAGTTCATGCAGCCCATGCTCACGTCATATCATGAGCTCTGAATCCAATAAGAGATGGAACCGAGTTGAATGTACTCCGGAACCAGCCACGGCAAGCACATTT MD:Z:6A5^GTTT20T16C6C1C1GA11A4^GT2A6 NH:i:1 HI:i:1 NM:i:18 SM:i:40 XQ:i:40 X2:i:0 XO:Z:UU XS:A:- XG:Z:M

Thanks for you help.

hartleys commented 6 years ago

Hmm. The gmap SAM file looks ok.

Can you give me the command line used with that gmap run?

On Feb 26, 2018 8:17 PM, "ehhill" notifications@github.com wrote:

That's odd, it should be aligned to the genome; it was from a minimap2 spliced alignment to the genome. Regardless, I've tried other sam files from gmap and these throw this error:

Error info: Exception in thread "main" java.lang.UnsupportedOperationException: empty.min at scala.collection.TraversableOnce.min(TraversableOnce.scala:222) at scala.collection.TraversableOnce.min$(TraversableOnce.scala:220) at scala.collection.mutable.ArrayOps$ofByte.min(ArrayOps.scala:203) at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3( commonSeqUtils.scala:710) at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3$ adapted(commonSeqUtils.scala:709) at scala.collection.TraversableLike.$anonfun$map$ 1(TraversableLike.scala:234) at scala.collection.immutable.List.foreach(List.scala:389) at scala.collection.TraversableLike.map(TraversableLike.scala:234) at scala.collection.TraversableLike.map$(TraversableLike.scala:227) at scala.collection.immutable.List.map(List.scala:295) at internalUtils.commonSeqUtils$.initSamRecordIterator( commonSeqUtils.scala:709) at qcUtils.runAllQC$.runOnSeqFile(runAllQC.scala:1020) at qcUtils.runAllQC$.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)

Here is an entry in this sam file. 1_377103 16 CS10_Chromosome_05 746131 40 12S5M1I7M4D54M1I7M1I5M2D9M24S 0 0 CTGATTCGCCGTGACTCTTTGCTTAGTTCATGCAGCCCATGCTCACGTCATATCATGAGC TCTGAATCCAATAAGAGATGGAACCGAGTTGAATGTACTCCGGAACCAGCCACGGCAAGCACATTT MD:Z:6A5^GTTT20T16C6C1C1GA11A4^GT2A6 NH:i:1 HI:i:1 NM:i:18 SM:i:40 XQ:i:40 X2:i:0 XO:Z:UU XS:A:- XG:Z:M

Thanks for you help.

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ehhill commented 6 years ago

I've generated a new gmap file using gmap -D ~/MinION/allReads/alignment_gmap/ -d CS10_db --nofails -f samse --min-intronlength=10 --max-intronlength-middle=1000 --max-intronlength-ends=1000 AllReads_renamed.fasta

Then used samtools/bamtools to convert to sorted .BAM.

This is the .SAM file: 1_9 16 CS10_Chromosome_04 356657 3 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 0 0 TCCCTCCCCAAAAGCAAAGTGTAGCGAGAGTTCATGCGACTGGGGTTTCCATAACCGAACCTCGCATTTCTGAGCGATGCAAAGGTGTTCCTGCGGGAAGATTCTCCCATGCACTCAAAAGAAGATAATAAAGCAAATATTCTTCTGCCAGTTGGTCTTTAATGACGACGGCTGCCGATCATCTTCGCATCAATATACCCAAGCGCCGCCGCGTCGTAGGCAAACAATGGAGAATCTATATGAACGCCGTCCATGCTGTTGGTGGCGATCGCATCGATTGCGTCAATAGTCTGGTTTCATACGCTTCATGCCCATATCCTACCGCTCAAAAGATGCATGGTGCAGGCCCTCGAAGGTCAGTGGTGACAGCCGCCGTATAAAGGACGCCCCTGGTAAAAGCATGACTCATTGGGGCGTTTCCACCCACCAGGTGTCGTCTCTACTCAAACAGCCCTCGCTACTGAACGCGAGGGTCCTATGACTACAGTTATCTCAATGTTTCTGGCGTATGACGCTATCTATTGCGCACGCCGCGTAAAATAATACGGCTGGACGTAAATCAGTGAGGCCTGGTGCTTTACCCGGCGCCGACTGGAAATACCAGCAGTACGCGCGGCGGAGAGTACAATAAGGAAGAAAAGATTGAGCGTAAGGTAACGGTAGAGCGGCGCCAGGAATACATGCGGGTCTTAAGGATTGACGTCGGTGATTGCGCTAACGTCCGCGGTTAGATCCATTGCAGGATCAGGCGCTTGATCGGGGTCCTGCCCCAATAGTCCTACACCAAGCGCGTCGTTAGACTTCCCTTTGGTACAATAATCACCTCTTGCCGCATCAGTGGTACGCCCACTATCTACTCTGCCGGACTTTTTCTGGCAATATTCCAGCAGTAACCTACCAGGCGCACAAAGGCGAAAGTGGCCTTTTAGTTCTTCCGCATTTCCATTTAGACGCATCAGTACTCCGGTAGACTTCAAATGGCGTTGCTTCCTTCAGCTCTATCTCCCAGTTTCTCCACAGGCAAATGAGCGCATATTGCGCCCTTTCTGACCCCGGCGATATCATCGCTGATCGTAAGCCGGAAGGCGATAAACGAGGTGACACCATCATGAACCGCATTTCCAAAGCCGATAAGCAAGTTTCTTTTCCGATCCTTTTGCATCAATGGGAGGCGTTCTGTTTTCGTTACTGTTGCCTCGAGAGCATCCCCAACGTTCTATTGATTTGGTTCCCTTTACTCTCCCCAAACTATCATTCATCAACATTGCTTTTCCCTTGTCCCCGCTACCGACAACGACATCAGATCCACTTCTATCCCCATATCATCACTTAGAATCTGGTTCAATCTTTTTCGTTAATTTTTCTTCAAAGGCGATATGACTTTACGCCAATGTCCTGATCCAGGAGTGCTTTCTTCTGCATTTCGGTTAAAGGCGCGTAAACGGGGTTGCATGGGCGGGCGGGAATTTCCATGCCATCAATTTGGCGCGAACCATACATCCAGGTTCAACTCAATACTATCGGCGTTGATCTTCCATCACACTTGCTCTTTTCTCATTCCGGATTCAATGGAGTTTTTGGCACCAGAGCGGGTTCAGAATATTGCCCGCCACCACAACCGGTACGCCAACATCATGCATGCTTATACCTCCGCCATACAAACCGTTTGTTCCGTTAGAGTTGTGCGAAAATCGGGTTAAAACAATCTGGAACGAACTGGAACGAAGCATCAA MD:Z:5^G6^TTCA1G1T2^TC0A15^CT0A9A1ATC3^AA0G4T11C1T7^TC45AT2A10^GTGT1G5^CTGC0C1C7^AACATC2C4^GCT18C15C1C3G11CA1AA1CA1T2CT16C5^ACA1CCC5T1^CCAT4TA1T6CC2A11CA12^G6^C0A2AC2G2^A0T4GT1^GCTC10^T2A2GC21C20C16A1^A0C2G15GG5T2C10C3G5^TGCG0GA2T2C16AT1C5CC5C5C10CT1A1CC3T1C2^A2A2A1CGCC11T8C4C3^GGTGTAGA16^G1C9C4TTA1^A7C3^GCG0TA1T7A12GTGA1CA2C2T6AT13CTA2CA11C14C3ACCA19CAC5TC1GC1TA1C4C1G18AT6^C2TA10C3T1^C1T6TTA2G13T3TC2T5^TTC20C2A15^AAAGG6^T3T2^CTTTTTT5TC9^TTTCTA2T2^T6^TG1C1CA6CACA3TC2TC3T2T6T1CT1T5A1^G0GG5C5^CTAGGC6C28T2^GGGG0G15T10CC9^TTG1T13T2T1T9TG3TAAT5T15TC9C2CCA1^G4T3G3TT1A6^AC15G21C11TT2C1CAGAG1CC1TTTC7ACAC4TA1T1^TCCT1TC2C8GAG9A1ACAC11^GG8G12^TCTTC0C9T4C12^TA0GC6TT2CT25^TACT7C2G6T2CTGA4^CC2CC18T8T1A9T1TA1T12T10^CCACCACA1A4T7T8 NH:i:1 HI:i:1 NM:i:543 SM:i:40 XQ:i:40 X2:i:0 XO:Z:UU XS:A:+ XG:Z:M 1_11 0 CS10_Chromosome_02 2151720 40 640S14M1I3M1I19M2D24M5I26M1I22M5D15M2I13M3D4M1D8M6S 0 0 TTGGTATACTTCGTTATCGGAGATTGATGGTTTGTTGGTGCGCTGATGTTGTAGGGGCTTGTGCGCTACTCAGACACCGGGTGCATCGCTTTGCAAACTCTTTCATTGTGAAGGCTGAACTGCATCTGATGGATTTGTTTCCTGGTGAACCGATTCAGCGGGTCCACTTTTATCACAGCACCTAGCCATTCCCGATAACCTTTCGGTTGAAGGAAGAAAGAAAACTTTGGATCGGAGGAGAAAATGACAAAAGAAATTACAGATAACGGCTGGATATTGCAGGTACAGCGAGAGGCGGAGGCGGCAGCGGATATGACGGCGAAAAGCCTCCTGCCAGCCAGTACTGGTGAAGCGGACAGAGACTTACCTACCTCAGATTTATATCCAGGCGGATTTTACGGAGGCGGATACGATACCTCTGGACCTCAATTTAAGCATTTTCGTCAAACTCAGCCGCACAACCAAGGCTACCAGAATGAAATTACTGGCGGAAATTTGGCCAGAGAATTGGGCACGTGGTAACATAAACCGACGTATCGCTGGTACGGCAGCAGGAGACTCAGCGTACTAAGGCAGCAAATCAGTCTGCAGTACCCTGCCAGCTTATCATTATGCGGAACCCTCCGCCAGCATAACTAAACCACAAGACCCAGGAACGCCCGTTGGAGACCGATGGGAAGGTTAAGCCTGCAAAGAAAGAAGGAAGAAGGAAGAGCGACGCCGCTGGGAGGAAGGAAGAGAACCGCCGTCGTAAATGGCGCCGCCGCCGCCATAGCGCCGGTACAGCCGCCATCAGTCCACGCA MD:Z:12A23^CG2CAC2AGAAAAG11A29AG1A13^ATGGC24G1C1^GCT0T3^A8 NH:i:2 HI:i:1 NM:i:39 SM:i:40 XQ:i:40 X2:i:0 XO:Z:UM XS:A:+ XG:Z:M

I run QoRTs with: java -Xmx4G -jar QoRTs.jar QC --singleEnded --generatePlots allreads.gmap.new.sam EVM6.gtf ~/coverage_plot/test

And get the error message:

Checking first 10000 reads. Checking SAM file for formatting errors... ============================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.UnsupportedOperationException: empty.min at scala.collection.TraversableOnce.min(TraversableOnce.scala:222) at scala.collection.TraversableOnce.min$(TraversableOnce.scala:220) at scala.collection.mutable.ArrayOps$ofByte.min(ArrayOps.scala:203) at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3(commonSeqUtils.scala:710) at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3$adapted(commonSeqUtils.scala:709) at scala.collection.TraversableLike.$anonfun$map$1(TraversableLike.scala:234) at scala.collection.immutable.List.foreach(List.scala:389) at scala.collection.TraversableLike.map(TraversableLike.scala:234) at scala.collection.TraversableLike.map$(TraversableLike.scala:227) at scala.collection.immutable.List.map(List.scala:295) at internalUtils.commonSeqUtils$.initSamRecordIterator(commonSeqUtils.scala:709) at qcUtils.runAllQC$.runOnSeqFile(runAllQC.scala:1020) 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)

jackhump commented 5 years ago

HI @hartleys , thanks for writing such a useful tool. I'm also trying to apply QoRTs to long read sequencing data generated by PacBio IsoSeq3 and aligned to the human genome with minimap2. My minimap parameters were:

minimap2 -ax splice -uf -C5 hg38.fa {sample}_hq_transcripts.fq > {sample}_hq_transcripts.sam

I've tried running QoRTs on both the SAM file and the coordinate sorted and indexed BAM file created by samtools.

This is the command I've been trying:

/share/apps/java/bin/java -jar /SAN/vyplab/HuRNASeq/QoRTs-STABLE.jar QC\
    --singleEnded \
    --stranded \
    --verbose \
    --keepMultiMapped \
    --maxReadLength 10000 \
    --minMAPQ 0 \
    aligned/{sample}_transcripts.sam \
    gencode.v30.annotation.gtf \
    ../test/

I gradually added all the extra flags in vain to get it to run.

This is the error message I got each time:

Error info:
Exception in thread "main" java.lang.UnsupportedOperationException: empty.min
    at scala.collection.TraversableOnce.min(TraversableOnce.scala:222)
    at scala.collection.TraversableOnce.min$(TraversableOnce.scala:220)
    at scala.collection.mutable.ArrayOps$ofByte.min(ArrayOps.scala:203)
    at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3(commonSeqUtils.scala:710)
    at internalUtils.commonSeqUtils$.$anonfun$initSamRecordIterator$3$adapted(commonSeqUtils.scala:709)
    at scala.collection.TraversableLike.$anonfun$map$1(TraversableLike.scala:234)
    at scala.collection.immutable.List.foreach(List.scala:389)
    at scala.collection.TraversableLike.map(TraversableLike.scala:234)
    at scala.collection.TraversableLike.map$(TraversableLike.scala:227)
    at scala.collection.immutable.List.map(List.scala:295)
    at internalUtils.commonSeqUtils$.initSamRecordIterator(commonSeqUtils.scala:709)
    at qcUtils.runAllQC$.runOnSeqFile(runAllQC.scala:1031)
    at qcUtils.runAllQC$.run(runAllQC.scala:970)
    at qcUtils.runAllQC$allQC_runner.run(runAllQC.scala:680)
    at runner.runner$.main(runner.scala:98)
    at runner.runner.main(runner.scala)

For good measure here is a read from my SAM file:

TD_00712_Bha_Adr_MNP_Iso_BS62H9_STD_HQ_transcript/2666  0   chr16   57628620    60  183M21450N99M848N423M1580N133M650N148M1265N132M345N117M233N46M242N104M755N119M1921N269M1086N109M820N269M1486N1572M32S   *   0   0   CTCCCTCTCCGCACTAGCTGTCTGCCCTGCCCTGCCGTAGGAGATGGGCTGGGAGCCTCCCACGCTCTCCAGCTCACTCGGCAGGCAGCGGGGACCAGGGCTGGCAGGTTAAGCCTCTGGGGGTGGATCCTGAAAGGTGGTCCAGCCGCCTGGCCCTGCGTGGGACCCTCCACCTGGCAGCAGGTGGTGACTTCCAAGAGTGACTCCGTCGGAGGAAAATGACTCCCCAGTCGCTGCTGCAGACGACACTGTTCCTGCTGAGTCTGCTCTTCCTGGTCCAAGGTGCCCACGGCAGGGGCCACAGGGAAGACTTTCGCTTCTGCAGCCAGCGGAACCAGACACACAGGAGCAGCCTCCACTACAAACCCACACCAGACCTGCGCATCTCCATCGAGAACTCCGAAGAGGCCCTCACAGTCCATGCCCCTTTCCCTGCAGCCCACCCTGCTTCCCGATCCTTCCCTGACCCCAGGGGCCTCTACCACTTCTGCCTCTACTGGAACCGACATGCTGGGAGATTACATCTTCTCTATGGCAAGCGTGACTTCTTGCTGAGTGACAAAGCCTCTAGCCTCCTCTGCTTCCAGCACCAGGAGGAGAGCCTGGCTCAGGGCCCCCCGCTGTTAGCCACTTCTGTCACCTCCTGGTGGAGCCCTCAGAACATCAGCCTGCCCAGTGCCGCCAGCTTCACCTTCTCCTTCCACAGTCCTCCCCACACGGCCGCTCACAATGCCTCGGTGGACATGTGCGAGCTCAAAAGGGACCTCCAGCTGCTCAGCCAGTTCCTGAAGCATCCCCAGAAGGCCTCAAGGAGGCCCTCGGCTGCCCCCGCCAGCCAGCAGTTGCAGAGCCTGGAGTCGAAACTGACCTCTGTGAGATTCATGGGGGACATGGTGTCCTTCGAGGAGGACCGGATCAACGCCACGGTGTGGAAGCTCCAGCCCACAGCCGGCCTCCAGGACCTGCACATCCACTCCCGGCAGGAGGAGGAGCAGAGCGAGATCATGGAGTACTCGGTGCTGCTGCCTCGAACACTCTTCCAGAGGACGAAAGGCCGGAGGGGGGAGGCTGAGAAGAGACTCCTCCTGGTGGACTTCAGCAGCCAAGCCCTGTTCCAGGACAAGAATTCCAGCCAAGTCCTGGGTGAGAAGGTCTTGGGGATTGTGGTACAGAACACCAAAGTAGCCAACCTCACGGAGCCCGTGGTGCTCACCTTCCAGCACCAGCTACAGCCGAAGAATGTGACTCTGCAATGTGTGTTCTGGGTTGAAGACCCCACATTGAGCAGCCCGGGGCATTGGAGCAGTGCTGGGTGTGAGACCGTCAGGAGAGAAACCCAAACATCCTGCTTCTGCAACCACTTGACCTACTTTGCAGTGCTGATGGTCTCCTCGGTGGAGGTGGACGCCGTGCACAAGCACTACCTGAGCCTCCTCTCCTACGTGGGCTGTGTCGTCTCTGCCCTGGCCTGCCTTGTCACCATTGCCGCCTACCTCTGCTCCAGGAGGAAACCTCGGGACTACACCATCAAGGTGCACATGAACCTGCTGCTGGCCGTCTTCCTGCTGGACACGAGCTTCCTGCTCAGCGAGCCGGTGGCCCTGACAGGCTCTGAGGCTGGCTGCCGAGCCAGTGCCATCTTCCTGCACTTCTCCCTGCTCACCTGCCTTTCCTGGATGGGCCTCGAGGGGTACAACCTCTACCGACTCGTGGTGGAGGTCTTTGGCACCTATGTCCCTGGCTACCTACTCAAGCTGAGCGCCATGGGCTGGGGCTTCCCCATCTTTCTGGTGACGCTGGTGGCCCTGGTGGATGTGGACAACTATGGCCCCATCATCTTGGCTGTGCATAGGACTCCAGAGGGCGTCATCTACCCTTCCATGTGCTGGATCCGGGACTCCCTGGTCAGCTACATCACCAACCTGGGCCTCTTCAGCCTGGTGTTTCTGTTCAACATGGCCATGCTAGCCACCATGGTGGTGCAGATCCTGCGGCTGCGCCCCCACACCCAAAAGTGGTCACATGTGCTGACACTGCTGGGCCTCAGCCTGGTCCTTGGCCTGCCCTGGGCCTTGATCTTCTTCTCCTTTGCTTCTGGCACCTTCCAGCTTGTCGTCCTCTACCTTTTCAGCATCATCACCTCCTTCCAAGGCTTCCTCATCTTCATCTGGTACTGGTCCATGCGGCTGCAGGCCCGGGGTGGCCCCTCCCCTCTGAAGAGCAACTCAGACAGCGCCAGGCTCCCCATCAGCTCGGGCAGCACCTCGTCCAGCCGCATCTAGGCCTCCAGCCCACCTGCCCATGTGATGAAGCAGAGATGCGGCCTCGTCGCACACTGCCTGTGGCCCCCGAGCCCGGCCCAGCCCCAGGCCAGTCAGCCGCAGACTTTGGAAAGCCCAACGACCATGGAGAGATGGGCCGTTGCCATGGTGGACGGACTCCCGGGCTGGGCTTTTGAATTGGCCTTGGGGACTACTCGGCTCTCACTCAGCTCCCACGGGACTCAGAAGTGCGCCGCCATGCTGCCTAGGGTACTGTCCCCACATCTGTCCCAACCCAGCTGGAGGCCTGGTCTCTCCTTACAACCCCTGGGCCCAGCCCTCATTGCTGGGGGCCAGGCCTTGGATCTTGAGGGTCTGGCACATCCTTAATCCTGTGCCCCTGCCTGGGACAGAAATGTGGCTCCAGTTGCTCTGTCTCTCGTGGTCACCCTGAGGGCACTCTGCATCCTCTGTCATTTTAACCTCAGGTGGCACCCAGGGCGAATGGGGCCCAGGGCAGACCTTCAGGGCCAGAGCCCTGGCGGAGGAGAGGCCCTTTGCCAGGAGCATAGCAGCAGCTCGCCTACCTCTGAGCCCAGGCCCCCTCCCTCCCTCAGCCCCCCAGTCCTCCCTCCATCTTCCCTGGGGTTCTCCTCCTCTCCCAGGGCCTCCTTGCTCCTTCGTTCACAGCTGGGGGTCCCCGATTCCAATGCTGTTTTTTGGGGAGTGGTTTCCAGGAGCTGCCTGGTGTCTGCTGTAAATGTTTGTCTACTGCACAAGCCTCGGCCTGCCCCTGAGCCAGGCTCGGTACCGATGCGTGGGCTGGGCTAGGTCCCTCTGTCCATCTGGGCCTTTGTATGAGCTGCATTGCCCTTGCTCACCCTGACCAAGCACACGCCTCAGAGGGGCCCTCAGCCTCTCCTGAAGCCCTCTTGTGGCAAGAACTGTGGACCATGCCAGTCCCGTCTGGTTTCCATCCCACCACTCCAAGGACTGAGACTGACCTCCTCTGGTGACACTGGCCTAGGGCCTGACACTCTCCTAAGAGGTTCTCTCCAAGCCCCCAAATAGCTCCAGGCGCCCTCGGCCGCCCATCATGGTTAATTCTGTCCAACAAACACACACGGGTAGATTGCTGGCCTGTTGTAGGTGGTAGGGACACAGATGACCGACCTGGTCACTCCTCCTGCCAACATTCAGTCTGGTATGTGAGGCGTGCGTGAAGCAAGAACTCCTGGAGCTACAGGGACAGGGAGCCATCATTCCTGCCTGGGAATCCTGGAAGACTTCCTGCAGGAGTCAGCGTTCAATCTTGACCTTGAAGATGGGAAGGATGTTCTTTTTACGTACCAATTCTTTTGTCTTTTGATATTAAAAAGAAGTACATGTTCATTGTAGAGAATTTGGAAACTGTAGAAGAGAATCAAGAAGAAAAATAAAAATCAGCTGTTGTAATCACCTAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAAA SJ?fs_]iZ|i~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~h~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~2~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~ NM:i:4  ms:i:3711   AS:i:3288   nn:i:0  ts:A:+  tp:A:P  cm:i:1200   s1:i:3568   s2:i:0  de:f:0.0011 rl:i:37

Thanks again!

bwlang commented 3 years ago

I'm seeing the same issue on the attached bam: ds.bam.zip run like this:

qorts QC --singleEnded ds.bam gencode.v35.primary_assembly.annotation.gtf .