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 that 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 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 CDS_123.cds.gtf --genomeFastaFiles Rs.R1_R9.fasta
1st read mapping
/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:
/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
I want to ask you that If I have many samples, How I can create one common set of novel junctions for all samples by merging them. Then you generate a new genome using annotated junctions and the common set of novel junctions, and re-run all the samples with this new genome - this would be the 2-nd pass