Question: STAR BAM into RSEM
1
gravatar for Biogeek
3.1 years ago by
Biogeek350
Biogeek350 wrote:

Hi,

Usually when I used RSEM I feed my left reads and right reads in as .fastq files. Left is the forward and right is the reverse. I took genome files created an index in STAR and uses quantmode so that I can put the .BAM out file into RSEM.

In the STAR paper it says "--paired-end --forward-prob 0 options are applicable to paired stranded RNA-seq data such as Illumina stranded Tru-seq protocol. Refer to RSEM documentation for detailed description of RSEM parameters."

When I refer to the RSEM manual, it gives this -

----------------
--forward-prob <double>
Probability of generating a read from the forward strand of a transcript. Set to 1 for a strand-specific protocol where all (upstream) reads are derived from the forward strand, 0 for a strand-specific protocol where all (upstream) read are derived from the reverse strand, or 0.5 for a non-strand-specific protocol. (Default: 0.5)
----------------------

We used Illumina sequencing, however forward reads are always left and reverse reads are always left.

This has left me confused. Can someone explain this to me kindly?

Thanks.

rsem star forward-prob • 1.9k views
ADD COMMENTlink modified 3.1 years ago by Devon Ryan92k • written 3.1 years ago by Biogeek350
2
gravatar for Devon Ryan
3.1 years ago by
Devon Ryan92k
Freiburg, Germany
Devon Ryan92k wrote:

The explanation given for the --forward-prob option in RSEM is incredibly opaque and should just be ignored. The description given by STAR is sufficient. If you have strand-specific data then use --forward-prob 0. If you're unsure, then use the infer_experiment function from RSeQC and post the output.

ADD COMMENTlink written 3.1 years ago by Devon Ryan92k

Thanks.

So for Illumina hiseq with truseq chemistry, we know the sense strand is the 5-3' (left: forward) and the antisense is 3-5' ( right/ reverse) read from knowledge. This is paired end with strand specific. So I'm asumming I still use an integer of 0 for this flag?

ADD REPLYlink written 3.1 years ago by Biogeek350
1

A stranded dataset gets a value of 0, so yes.

ADD REPLYlink modified 3.1 years ago • written 3.1 years ago by Devon Ryan92k

Thanks Devon. Just wanted to make sure. I agree with you though, the manual lacks Clarity.

ADD REPLYlink written 3.1 years ago by Biogeek350
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