Interesting. We wrap all aligners, including tophat, with MergeBamAlignment
since aligners tend to produce inconsistent output. This is coming from a
recovering aligner author. Can you try that?
Also you used TopHat, so you could have the original FASTQs that you provided
TopHat.
Nils
Thumb typed for added typos
> On Sep 5, 2014, at 10:18 AM, "Trakhtenberg, Feliks"
> <[email protected]> wrote:
>
> 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]> 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]> 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 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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