STAR-2 pass mapping

YogeshYogesh south koreaMember

I want to use STAR 2-pass alignment steps for SNP detection in RNAseq data:

But I am getting very confused, I using STAR 2.5.3a version:

I can understand the there 4 steps need to perform in STAR 2- pass mapping.

1) 1st Genome generator

2) ButI can't able to understand how to run 1st pass aligner for all sample together or separately.

3) Genome generator again.

4) After 1st pass aligner how to specify all tab files in 2nd aligner, what should be the parameter to filter the SJ.out. tab files need to be considered? how to prefix the SJ.out.tab with the different name?

Command line which I am using to perform all four steps:

1) 1st Genome Generator

/STAR --runThreadN 6 --runMode genomeGenerate --genomeDir /data/SNU_work/genome --sjdbGTFfile Radish_123.cds.gtf --genomeFastaFiles Rs.R1_R9.fasta

1st read mapping

2) /home/yog/software/STAR-2.5.3a/source/STAR --genomeDir /data/SNU_work/genome --readFilesIn 216_R1.fq 216_R2.fq --runThreadN 6

2nd Genome generator:

3) /STAR --runThreadN 6 --runMode genomeGenerate --genomeDir /data/SNU_work/genome --sjdbOverhang 124 --sjdbFileChrStartEnd /data/SNU_work/SJ.out.tab --genomeFastaFiles Rs.R1_R9.fasta

Now here I am confused how to generator all Sj.out.tab altogether or should generator one by one but how to mention different name according to RNAseq library?

4) again star aligner

Please look into command line also and suggest if I am making all correct or not.

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