Hi ulz_peter and everybody,
I have a problem when I try to execute the next command (step in my exome analysis):
java -Xmx4g -Djava.io.tmpdir=/tmp \
-jar picard/SortSam.jar \
SO=coordinate \
INPUT=input.sam \
OUTPUT=output.bam \
VALIDATION_STRINGENCY=LENIENT \
CREATE_INDEX=true
The output to this (above)is a error message: It doesn´t find or load the jarfile SamSort.jar.Also ,sometimes, the error message is : it has not loaded the main class (or similar).
But I have seen the SortSam.jar file inside my picards-tools folder.I have downloaded well the picard-tools with SortSam.jar included.I have tried with differents paths for SamSort.jar,but the problem is the same.
What could I do?.Somebody could help me,please, to go on with my exome analysis?
Waiting for your answer,Thank you so much .
JM
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Thank you for the nice how-to guide.
I have a couple questions about it.
1. The second step of 2.2 Actual Alignment uses the -f flag. Is this to specify what the output .sai file is called? I've looked through the bwa manual page (http://bio-bwa.sourceforge.net/bwa.shtml) and it doesn't mention -f.
2. This question is about the -r flag that is used on the same command and the next. The guide has:
I don't know how much it matters, but should there be "@RG" instead of "@RQ" after the open quotes?Code:bwa sampe -f out.sam -r "@RQ\tID:<ID>\tLB:<LIBRARY_NAME>\tSM:<SAMPLE_NAME>\tPL:ILLUMINA" hg19 input1.sai input2.sai input1.fq input2.fq
Thanks,
BlakeLeave a comment:
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Hi, I need some advice regarding the downstream analysis and calling of the variants , I am trying to establish a pipeline for my lab and am new to exome sequecing data analysis. I am now trying to use it with a single sample(paired end) from the 1000 genome project. Sample 96 to be precise. I have already done the alignment and also done till the indexing of the sorted bam file. So the next step would be using the GATK tool
and identify the target regions for realignment then realign the BAM to get better INDEL calling and then calling the different packages of GATK to call the SNP and INDELs, I want to ask , I downloaded the latest version of GATK, is it advisable to work with that or with older versions? also in some forums I see the MarkDuplicates step is skipped and since the realignment is done with the GATK again for better INDEL calling , it seems we can skip this step. Suggestions should be welcome.Leave a comment:
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Need some suggestions for downstream analysis
Hi, I need some advice regarding the downstream analysis and calling of the variants , I am trying to establish a pipeline for my lab and am new to exome sequecing data analysis. I am now trying to use it with a single sample(paired end) from the 1000 genome project. Sample 96 to be precise. I have already done the alignment and also done till the indexing of the sorted bam file. So the next step would be using the GATK tool
and identify the target regions for realignment then realign the BAM to get better INDEL calling and then calling the different packages of GATK to call the SNP and INDELs, I want to ask , I downloaded the latest version of GATK, is it advisable to work with that or with older versions? also in some forums I see the MarkDuplicates step is skipped and since the realignment is done with the GATK again for better INDEL calling , it seems we can skip this step. Suggestions should be welcome.Leave a comment:
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new to exome analysis what software
Hi Guys
I am new to exome analysis and was hoping for guidance as to what software is accepted as the most robust pipeline for finding SNPs
cheers
JulianLeave a comment:
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I think it's important add in manual and in the wiki to add that vcf file, hg19.fasta
are in GATK bundle to which it's possible to access with an ftp client:
GATK budle ftp with an ftp client
http://gatkforums.broadinstitute.org...lic-ftp-server
I think that it's an important step to add in wiki
http://seqanswers.com/wiki/How-to/exome_analysisLeave a comment:
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Hi you all and congratulations for this useful thread.
I am trying to reproduce the pipeline posted at the beginning of the post as an alternative way for SNP analysis.
I am not actually experienced in NGS but I have to deal with the results of exome analyses coming from MiSeq sequencer and I would like to improve (compare) the results obtained trhough the MiSeq machine (BWA, CASAVA...).
According to the tutorial posted by ulz_peter (thanks again) I have performed the initial reference genome indexing (hg19) with the last updated version of bwa (0.6.2) and I obtained 5 different files as a result
hg19.amb
hg19.ann
hg19.bwt
hg19.pac
hg19.sa
According to other threads (http://seqanswers.com/forums/showthread.php?t=20705) it seems that the expected number of resulting files is 8. May I continue with this five files or it should be better to work with an earlier version of bwa? just in order to be able to reproduce the pipeline here described.
On the other hand, and thinking on the next step in the pipe, according to the BWA alignment options suggested in the tutorial:
"the -I option tells BWA to use Illumina1.3+ qualities"
but if I am not misunderstood, Miseq fastq results are in Sanger format (Illumina 1.8+), so may I use the -I option or not?
I think I am asking for very basic things but you know, basic knowledge is crucial to understand complexity. So I´ll be grateful if anuone could help me. I promise to continue asking when I have a doubt.
Thanks in advanceLeave a comment:
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I get the following error when using GATK to perform local realignment around indels.
Anyone an idea what went wrong?
Code:E:\EXOME DATA ANALYSIS\1 Unzipped fastq>java -jar GenomeAnalysisTK.jar -T Realig nerTargetCreator -R hg19.fa -o Ot2363.bam.list -I Ot2363.marked.bam INFO 13:45:07,701 HelpFormatter - --------------------------------------------- ----------------------------------- INFO 13:45:07,710 HelpFormatter - The Genome Analysis Toolkit (GATK) v2.1-9-gb9 0951c, Compiled 2012/09/19 21:18:53 INFO 13:45:07,710 HelpFormatter - Copyright (c) 2010 The Broad Institute INFO 13:45:07,710 HelpFormatter - For support and documentation go to http://ww w.broadinstitute.org/gatk INFO 13:45:07,712 HelpFormatter - Program Args: -T RealignerTargetCreator -R hg 19.fa -o Ot2363.bam.list -I Ot2363.marked.bam INFO 13:45:07,712 HelpFormatter - Date/Time: 2012/09/20 13:45:07 INFO 13:45:07,712 HelpFormatter - --------------------------------------------- ----------------------------------- INFO 13:45:07,713 HelpFormatter - --------------------------------------------- ----------------------------------- INFO 13:45:07,720 GenomeAnalysisEngine - Strictness is SILENT INFO 13:45:07,723 ReferenceDataSource - Index file E:\EXOME DATA ANALYSIS\1 Unz ipped fastq\hg19.fa.fai does not exist. Trying to create it now. PROGRESS UPDATE: file is 15 percent complete PROGRESS UPDATE: file is 28 percent complete PROGRESS UPDATE: file is 39 percent complete PROGRESS UPDATE: file is 54 percent complete PROGRESS UPDATE: file is 67 percent complete PROGRESS UPDATE: file is 77 percent complete PROGRESS UPDATE: file is 89 percent complete PROGRESS UPDATE: file is 99 percent complete ##### ERROR -------------------------------------------------------------------- ---------------------- ##### ERROR A USER ERROR has occurred (version 2.1-9-gb90951c): ##### ERROR The invalid arguments or inputs must be corrected before the GATK ca n proceed ##### ERROR Please do not post this error to the GATK forum ##### ERROR ##### ERROR See the documentation (rerun with -h) for this tool to view allowabl e command-line arguments. ##### ERROR Visit our website and forum for extensive documentation and answers to ##### ERROR commonly asked questions http://www.broadinstitute.org/gatk ##### ERROR ##### ERROR MESSAGE: Couldn't write file E:\EXOME DATA ANALYSIS\1 Unzipped fastq \hg19.fa.fai because exception The process cannot access the file because anothe r process has locked a portion of the file ##### ERROR -------------------------------------------------------------------- ----------------------
Leave a comment:
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Concerning the sam to bam conversion and suppression of PCR duplicates steps, are there any reason to prefer Picard to samtools?
I tried SortSam from Picard and it seems to take much more time than samtools view + samtools sort.
I think I will use samtools, but I would like to know if there are advantages when using Picard.
Thank youLeave a comment:
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We had some discussion on this in the GATK forum here. Maybe that is of interest to you.Originally posted by rahilsethi View PostI have additional question to raonyguimaraes's post
Does anyone know in details about GATK -dcov option in UnifiedGenotyper. I tried to look in GATK Manual but could not find much about it other than the following information:
-dcov [50 for 4x, 200 for >30x WGS or Whole exome]
in the link:
http://www.broadinstitute.org/gatk/g...Genotyper.html
Also if not specified what default value this option takes?
If you anyone knows about it could you please send me the link to the information resource?
Thanks in advance
cheers,
SophiaLeave a comment:
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I am wondering if the step of variant quality score recalibration, after the variant calling is still in use. If I remember well, I read somewhere that it was no more performed. In addition, in the publications that I read recently, this step is not mentioned. Do you know why it has been abandoned?
Or what was the interest in the first place to recalibrate the quality of the variant bases after the variant calling, since there was the quality score recalibration before variant calling
?
Leave a comment:
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