Question: Orientation of reads in .fastq files from RNA-Seq
0
gravatar for newbio17
9 months ago by
newbio1710
newbio1710 wrote:

I have R1.fastq and R2.fastq from RNA-Seq experiment. I'm currently trying to perform differential expression using Salmon and DESeq2.

Since the RNA-Seq was done using stranded library kit, it was under my assumption that R1.fastq contains forward reads and R2.fastq contains reverse reads.

So, I used the following command to run Salmon:

salmon quant -i <index> -l ISF -1 <R1.fastq> -2 <R2.fastq> -o <output> --gcBias

This resulted in poor mapping rate (~3%) and confused me at first since I saw no issues with the sequencing data (e.g. no rRNA contamination, etc...).

Instead, when I tried with below command, I was able to reach ~90% mapping rate:

salmon quant -i <index> -l ISR -1 <R1.fastq> -2 <R2.fastq> -o <output> --gcBias

From the documentation, F/R of option -l tells Salmon which strand a read is coming from given a .fastq file:

F: read1 comes from the forward strand and read2 comes from the reverse strand

R: read1 comes from the reverse strand and read2 comes from the forward strand

So does this mean that in my R1.fastq, there are reverse strand reads?

Thanks in advance.

rna-seq salmon • 600 views
ADD COMMENTlink modified 9 months ago by swbarnes25.9k • written 9 months ago by newbio1710
1

This page should help you with the read orientation.

ADD REPLYlink written 9 months ago by genomax69k

Thank you genomax. However, table seems a little bit confusing to me.

From what I'm able to understand, if library kit Illumina TruSeq Stranded Total RNA was used, then reads from reverse-strand belongs to 1st read strand? If this is correct, this is why I would have to use ISR instead of ISF?

ADD REPLYlink written 9 months ago by newbio1710
2
gravatar for swbarnes2
9 months ago by
swbarnes25.9k
United States
swbarnes25.9k wrote:

Since the RNA-Seq was done using stranded library kit, it was under my assumption that R1.fastq contains forward reads and R2.fastq contains reverse reads.

That assumption was wrong, as you can see with your own data. In Truseq standed kits, read 1 is in the reverse orientation.

ADD COMMENTlink written 9 months ago by swbarnes25.9k
Please log in to add an answer.

Help
Access

Use of this site constitutes acceptance of our User Agreement and Privacy Policy.
Powered by Biostar version 2.3.0
Traffic: 1462 users visited in the last hour