long read QC #57

ehhill · 2018-02-23T03:58:09Z

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 · 2018-02-26T04:36:22Z

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

…

On Feb 22, 2018 10:58 PM, "ehhill" ***@***.***> 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 — You are receiving this because you are subscribed to this thread. Reply to this email directly, view it on GitHub <#57>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ACwu7GLtj6TvEZTCx6cybqyxUNqLPAq_ks5tXjdSgaJpZM4SQWlT> .

ehhill · 2018-02-26T22:24:19Z

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 · 2018-02-26T22:34:03Z

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" ***@***.***> 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. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#57 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ACwu7Jh6MHngNAoQ58H7nkTpqZQ4VLKEks5tYy8TgaJpZM4SQWlT> .

ehhill · 2018-02-27T01:04:53Z

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 · 2018-02-27T01:06:23Z

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

…

On Feb 26, 2018 8:04 PM, "ehhill" ***@***.***> 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 <#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( *'))+-($&&#&)*((%)*(&&%& — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#57 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ACwu7LaV9QVMmAG7yb8TFWjz8dpP7SA7ks5tY1S2gaJpZM4SQWlT> .

ehhill · 2018-02-27T01:17:02Z

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 · 2018-02-27T01:24:55Z

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" ***@***.***> 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. — You are receiving this because you commented. Reply to this email directly, view it on GitHub <#57 (comment)>, or mute the thread <https://github.com/notifications/unsubscribe-auth/ACwu7IKx30AH23YkCL49jWgy09gBKGZIks5tY1ePgaJpZM4SQWlT> .

ehhill · 2018-02-28T00:52:24Z

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 · 2019-04-23T21:03:34Z

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~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~~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Thanks again!

bwlang · 2021-08-29T02:37:11Z

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 .

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long read QC #57

long read QC #57

ehhill commented Feb 23, 2018

hartleys commented Feb 26, 2018 via email

ehhill commented Feb 26, 2018

hartleys commented Feb 26, 2018 via email

ehhill commented Feb 27, 2018

hartleys commented Feb 27, 2018 via email

ehhill commented Feb 27, 2018

hartleys commented Feb 27, 2018 via email

ehhill commented Feb 28, 2018

jackhump commented Apr 23, 2019

bwlang commented Aug 29, 2021 •

edited

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long read QC #57

long read QC #57

Comments

ehhill commented Feb 23, 2018

hartleys commented Feb 26, 2018 via email

ehhill commented Feb 26, 2018

hartleys commented Feb 26, 2018 via email

ehhill commented Feb 27, 2018

hartleys commented Feb 27, 2018 via email

ehhill commented Feb 27, 2018

hartleys commented Feb 27, 2018 via email

ehhill commented Feb 28, 2018

jackhump commented Apr 23, 2019

bwlang commented Aug 29, 2021 • edited Loading

bwlang commented Aug 29, 2021 •

edited

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