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indel realingment in RNAseq reads

YogeshYogesh south koreaMember

I am Trying to find SNP in the transcriptome. I have finished the Split'N'Trim and reassign mapping qualities step. after this can I do indel realingment using these command: But sorted bam file is needed, can I sort the bam file using samtool:

/home/samtools/samtool sort dedup.bam >sorted.dedub.bam

Generate intervals of interest from sample alignments:
java -jar GenomeAnalysisTK.jar \
-T RealignerTargetCreator \
-nt 4 \
-R refgenome.fa \
-I **sorted.dedup.bam **\
-o realign.intervals

Realign (multiple sequence alignment):
java -jar GenomeAnalysisTK.jar \
-T IndelRealigner \
-R refgenome.fa\
-targetIntervals realign.intervals \
-I sample1.sorted.dedup.bam \
-o sample1.sorted.dedup.realigned.bam

Fix mate pair info in BAM (PICARD):
java -jar $PICARDDIR/picard.jar FixMateInformation \
INPUT=sample1.sorted.dedup.realigned.bam \
OUTPUT=sample1.sorted.dedup.realigned.fixmate.bam \
SO=coordinate \

Best Answers


  • YogeshYogesh south koreaMember

    Can you please suggest in case of HaplotypeCaller,why Indel realingment is not recommended ?what is the difference between Haptotypecaller and UnifiledGenotyper?

  • YogeshYogesh south koreaMember

    Thanks. after variant calling, I want to separate SNP and Indels. Which parameter should I use to remove false positive from SNP and Indels?

    I do not have know SNP database for my species, the only draft genome is available, how I can do base recalibration before calling variant using HaplotypeCaller?

  • shleeshlee CambridgeMember, Broadie ✭✭✭✭✭

    Hi @Yogesh,

    If you have decided you need BQSR, then you can bootstrap a database of known SNPs. This article talks about bootstrapping and the forum has multiple discussion threads that discuss some finer details. You can search the forum for these using the upper-right Search.

    For considerations and commands before and after VQSR, I'd like to refer you to the Variant Filtering workshop hands-on tutorial. You can find the most recent iteration of this tutorial, as well as the data bundle (for hands-on practice), under DAY 2 on this blogpost. The command in section 4.1 shows you how to select out just SNPs using SelectVariants. You can similarly select out INDELs. Remember that these options exclude MIXED type sites. Be sure to check your data after commands to ensure they are as you expect.

    Also see this post, this post and this doc. The last document has a number of links to other informative documents.

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