Strand specific pair end sequencing

Question

[Bioinfonext]

I have a strand-specific pair-end Illumina sequencing data. I am not able to understand which option to use in the different software. 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

Thanks

[iraun]

RSeQC infer_experiment.py

[Bioinfonext]

Thanks,

I getting error when running RSeQC script. please suggest why I am getting error:

[root@psgl software]# /home/yog/software/RSeQC-2.6.4/scripts/infer_experiment.py Traceback (most recent call last): File "/home/yog/software/RSeQC-2.6.4/scripts/infer_experiment.py", line 49, in <module> from bx.bitset import * ImportError: No module named bx.bitset

Is it related to installation. I am using CentOS platform.

Command line which I used for installation:

[root@psgl RSeQC-2.6.4]# python setup.py install

[h.mon]

pip install RSeQC should take care of installing other python modules required. You can do pip install bx now.

[Bioinfonext]

still getting error

[root@psgl software]# cd RSeQC-2.6.4/ [root@psgl RSeQC-2.6.4]# pip install bx bash: pip: command not found... [root@psgl RSeQC-2.6.4]#

[Bioinfonext]

Please suggest the library type:

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

Wheather is fr-firststrand --fr assume stranded library fr-secondstrand for StringTie

What about HISAT2 and Trinity?

Thanks

Answer 1

[h.mon]

Do you know the library prep kit? Was it Truseq stranded mRNA? If yes, it is a dUTP protocol, so it is fr-firststrand - read more here.

[Bioinfonext]

we use this kit for library preparation:

Ovation RNA-Seq Systems 1–16 for Model Organisms PART NOS. 0351 ARABIDOPSIS kit