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Old 05-22-2018, 08:13 AM   #1
Location: Chile

Join Date: Oct 2014
Posts: 12
Default Questions about small RNA-seq (single-end and strand-specific)

Hello everyone!
I'm new analyzing transcriptomic.
I have small RNA-seq data, single-end from Rat.
Initially, I mapped the data with Bowtie with next command:

bowtie -p 50 --mm -n 0 --best --strata -m 1 /media/stdata/bowtie1_index/rn6/rn6_bw1 -q $each_file\.fastq -S $each_file\_n0_m1.sam

** -n 0 and -m 1, because we are probing parameters.

After, I noticed that the kit to make library was "TruSeq® Small RNA Library".
Reading in the web, I found that:

"For example, the Illumina TruSeq small RNA sample preparation kit produces strand-specific libraries. The kit specifically selects the RNA species that have a monophosphate group at its 5′-end and a hydroxyl group at 3′-end, a typical structure for miRNAs."

Then, I think that bowtie also must have option for strand-specific.
Considering that the data is single-end and default is --fr, Must I need to add option for strand-specific?

If so, what options are they?
I found:

--fr/--rf/--ff -1, -2 mates align fw/rev, rev/fw, fw/fw (default: --fr)
--nofw/--norc do not align to forward/reverse-complement reference strand

But I have single end, so I think is necessary to use "--nofw/--norc", but I'm confused which one to choose.
Name_less_ is offline   Reply With Quote
Old 06-07-2018, 05:59 AM   #2
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Location: Brno, Czech republic

Join Date: Nov 2010
Posts: 1

Hi Name_less_,

You don't say if you do a genomic alignment to you map directly to a smallRNA database(s).
In case you do genomic alignment you should not use --nofw/--norc for the Bowtie alignment as you would disallow alignment to the - strand of the genome. Instead, you should use strand-specific counting afterward (for example in FeatureCounts option -s 1).
If you do database (miRBase, piRBase, ...) you should use the --nofw/--norc option because you assume the sequences in the database are already in the same direction as they could be found in the organism.
Opplatek is offline   Reply With Quote

single-end reads, srna, strand-specific

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