Question: finding mutations in rna-seq transcriptome
0
gravatar for genya35
2.3 years ago by
genya3530
genya3530 wrote:

Hello,

Please suggest bioinformatics tools to identify indel and point mutations in RNA-seq transcriptome.

Thanks

rna-seq • 1.1k views
ADD COMMENTlink modified 2.3 years ago by grant.hovhannisyan2.0k • written 2.3 years ago by genya3530
3
gravatar for jared.andrews07
2.3 years ago by
jared.andrews076.9k
Memphis, TN
jared.andrews076.9k wrote:

You can use same tools used for finding them in whole genome sequencing. Be aware that most of your reads are only coming from one strand the majority of the time with RNA-seq, so het/homozygous calls are a lot less reliable.

GATK is kind of the standard, and objectively one of the most accurate when used properly. It's also under continuous development and has tons of options. VarScan2 is also quite good, though it's no longer actively updated. Avoid the samtools/bcftools method that you'll see in a lot of tutorials - it does not do well with low frequency mutations and generally isn't as accurate.

ADD COMMENTlink written 2.3 years ago by jared.andrews076.9k

What do I use to convert fast-q RNA-seq to bam format?

ADD REPLYlink written 2.3 years ago by genya3530
3

Convert is not a correct word here:) You need to map the reads (fastq file) to the reference genome (or transcriptome), if available, to obtain the bam file. To do it you can use for example STAR or Hisat2 software.

ADD REPLYlink written 2.3 years ago by grant.hovhannisyan2.0k

Is it possible to use rna-star on usegalaxy.org to do the alignment. I don't see two-pass mode option? It's not clear on the website.

ADD REPLYlink written 2.3 years ago by genya3530

It seems the two-pass mode isn't available on Galaxy, but STAR isn't too hard to set up locally if you've got enough RAM. It's kind of a hog, so you want at least 32 GB for it to run.

ADD REPLYlink written 2.3 years ago by jared.andrews076.9k

@jared.andrews07 is VarScan2 same as VarScan version 2? Should I Split'N'Trim and reassign mapping qualities with SplitNCigarReads before running samtools to generate mpileup, and finally run VarScan in somatic mode? Thanks

ADD REPLYlink written 2.2 years ago by genya3530

Yes, they are the same, though I'd somewhat recommend GATK over it, particularly for somatic calling. I imagine following GATK's best practices probably wouldn't hurt, and yes, you'll have to run in somatic mode.

ADD REPLYlink written 2.2 years ago by jared.andrews076.9k
0
gravatar for grant.hovhannisyan
2.3 years ago by
grant.hovhannisyan2.0k wrote:

As already mentioned, you can use GATK. But variant calling based on RNAseq data has its tricks, so better follow the best practices of GATK specifically for RNAseq.

ADD COMMENTlink written 2.3 years ago by grant.hovhannisyan2.0k

@yelekley7 : example bash script (from start to end) for GATK best practices in variant calling for RNAseq is available here.

ADD REPLYlink written 2.3 years ago by cpad011214k

I forgot to mention, that I have both tumor and normal fastqs and looking for somatic mutations. Is GATK best practices still appropriate? Thanks

ADD REPLYlink written 2.2 years ago by genya3530

I guess you can. But replace the variant caller with tumor specific variant caller (better somatic variant callers such as mutect) and subsequent steps

ADD REPLYlink modified 2.2 years ago • written 2.2 years ago by cpad011214k
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