Strand specific pair end sequencing

[Yogesh Gupta]

I have a strand-specific pair-end Illumina sequencing data. I am not able to understand which option to use in these different softwares. For each library I have two files: R1 and R2 and I am not sure about forward strand and reverse strand.

I have mapped these R1 and R2 fq files to genome. Please suggest how should I determine which option should I use for Trinity, Hisat2, and StringTie.

Is there any tool to find information about strand-specific pair-end reads?

Trinity:

 --SS_lib_type <string>          :Strand-specific RNA-Seq read orientation.

#                                           if paired: RF or FR,

hISAT2-Paired-end:

  --fr/--rf/--ff     -1, -2 mates align fw/rev, rev/fw, fw/fw (--fr)

StringTie

 --rf assume stranded library fr-firststrand

 --fr assume stranded library fr-secondstrand

It will be a great help for me.

Thanks

Yogesh

[Adriana Romero]

Rf is dUTP method 

"All RNA-seq libraries we make are with Illumina Truseq stranded RNA-seq method which protocol could be found at illumina website. The method is using random hexamer to make 1st strand which will be sequenced. Strand specificity is achieved by replacing dTTP with dUTP in the Second Strand Marking Mix (SMM). The incorporation of dUTP in second strand synthesis effectively quenches the second strand during amplification, since the polymerase used in the assay will not incorporate past this nucleotide. " 

I believe you have to use RF

[Yogesh Gupta]

Hi, 

output result of RSeQC:

This is PairEnd Data

Fraction of reads failed to determine: 0.0196

Fraction of reads explained by "1++,1--,2+-,2-+": 0.9360

Fraction of reads explained by "1+-,1-+,2++,2--": 0.0444

 Trinity (RFor FR)?

Wheather is it fr-firststrand or  fr-secondstrand for StringTie?

What about HISAT2 ( --fr/--rf/-) and

Thanks

Yogesh