I just did my own RNAseq, analyzed with Tophat already optimized and used by
many at HMS, and tried to convert the resulting BAM to Fastq using Picard. It
seems like there is a large population of researchers who use BAM produced by
Tophat, so not sure how to go about it. thanks though.
________________________________
From: Nils Homer [[email protected]]
Sent: Friday, September 05, 2014 10:12 AM
To: Nils Homer
Cc: Trakhtenberg, Feliks; [email protected]
Subject: Re: [Samtools-help] Picard error Illegal Mate State in converting
Tophat BAM output to Fastq
You should also go to the folks who provided you the BAM and ask for them to
fix it, as this goes against common conventions and best practices.
Unfortunately Picard cannot support BAMs with such problems. I apologize for
your inconvenience.
N
Thumb typed for added typos
On Sep 5, 2014, at 9:33 AM, Nils Homer
<[email protected]<mailto:[email protected]>> wrote:
Hey Feliks,
it does indeed look a problem with your SAM file as described. Conceptually
the resolve pairs seems like a correct solution, and so I would contact the
author that script or leave report an issue on that repository's github website.
N
On Wed, Sep 3, 2014 at 9:47 AM, Trakhtenberg, Feliks
<[email protected]<mailto:[email protected]>>
wrote:
Hello, I want to use SamToFastq on BAM produced by Tophat. I am getting Illegal
Mate State error, details below. Setting VALIDATION_STRINGENCY to LENIENT or to
SILENT did not solve the issue, even if I first pre-filter to unique reads
using samtools view -bq 4 accepted_hits.bam > filtered.bam.
Same issue was reported here https://github.com/jeff-k/resolvepairs explaining
that the error is due to more than one pair of reads having the same query
name. The proposed solution was to add unique id to the names of reads
belonging to the same mate pair using the Resolvepair script, which failed for
me on line 94 due to syntax error. Would appreciate a solution, thank you.
Java version 1.7. Program report (Command line included below):
Job </opt/java/jdk1.7.0_45/bin/java -jar /opt/picard-tools-1.79/SamToFastq.jar
VALIDATION_STRINGENCY=SILENT INPUT=filtered_old.bam FASTQ=read_1.fastq
SECOND_END_FASTQ=read_2.fastq> was submitted from host <mezzanine.orchestra> by
user <ft42> in cluster <hms_orchestra>.
Job was executed on host(s) <clarinet002-164.orchestra>, in queue <mini>, as
user <ft42> in cluster <hms_orchestra>.
</home/ft42> was used as the home directory.
</groups/benowitz/BamToFastq> was used as the working directory.
Started at Wed Sep 3 09:31:24 2014
Results reported at Wed Sep 3 09:31:27 2014
Your job looked like:
------------------------------------------------------------
# LSBATCH: User input
/opt/java/jdk1.7.0_45/bin/java -jar /opt/picard-tools-1.79/SamToFastq.jar
VALIDATION_STRINGENCY=SILENT INPUT=filtered_old.bam FASTQ=read_1.fastq
SECOND_END_FASTQ=read_2.fastq
------------------------------------------------------------
Exited with exit code 1.
Resource usage summary:
CPU time : 1.36 sec.
The output (if any) follows:
[Wed Sep 03 09:31:26 EDT 2014] net.sf.picard.sam.SamToFastq
INPUT=filtered_old.bam FASTQ=read_1.fastq SECOND_END_FASTQ=read_2.fastq
VALIDATION_STRINGENCY=SILENT OUTPUT_PER_RG=false RE_REVERSE=true
INCLUDE_NON_PF_READS=false READ1_TRIM=0 READ2_TRIM=0
INCLUDE_NON_PRIMARY_ALIGNMENTS=false VERBOSITY=INFO QUIET=false
COMPRESSION_LEVEL=5 MAX_RECORDS_IN_RAM=500000 CREATE_INDEX=false
CREATE_MD5_FILE=false
[Wed Sep 03 09:31:26 EDT 2014] Executing as
ft42@clarinet002-164<mailto:ft42@clarinet002-164> on Linux
3.2.41-bpo.4-ritg1-amd64 amd64; Java HotSpot(TM) 64-Bit Server VM 1.7.0_45-b18;
Picard version: 1.79(1282)
[Wed Sep 03 09:31:27 EDT 2014] net.sf.picard.sam.SamToFastq done. Elapsed time:
0.01 minutes.
Runtime.totalMemory()=1140850688
FAQ: http://sourceforge.net/apps/mediawiki/picard/index.php?title=Main_Page
Exception in thread "main" net.sf.picard.PicardException: Illegal mate state:
HWI-ST1006:65:C0VRNACXX:2:1102:15430:43570
at net.sf.picard.sam.SamToFastq.assertPairedMates(SamToFastq.java:325)
at net.sf.picard.sam.SamToFastq.doWork(SamToFastq.java:146)
at
net.sf.picard.cmdline.CommandLineProgram.instanceMain(CommandLineProgram.java:177)
at net.sf.picard.sam.SamToFastq.main(SamToFastq.java:119)
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