Finding orientation of single-ended reads (R or F)

Question

[Moneeb Bajwa]

Hello,

How would I find out whether my single-ended reads are forward or reverse? Here are the SRA reads I used: https://www.ncbi.nlm.nih.gov/sra?linkname=bioproject_sra_all&from_uid=434667.

Thank you!

EDIT: Nevermind...the link tells with "spot descriptor"

[lieven.sterck]

what specifically do you want to know? if the read is from the forward or the reverse dna strand? if the read is forward or reverse in the sequencing protocol?

The former you can't obtain from the read itself, the latter should be always forward (the reverse one is the paired end mode)

[Moneeb Bajwa]

So would I put F (forward) for when Trinity asks for Ss_lib_type when I am aligning?

[h.mon]

If you want to know which setting to use with trinity:

1) map some reads with STAR (you will need some 32-34Gb memory). The index has to be built with an annotation built-in, or you can provide an annotation at run time. You don't need to output a bam file, just the gene counts:

  STAR --outFileNamePrefix temp. --genomeDir /path/to/STAR_index \
--outSAMtype None --readFilesIn file.fastq.gz --readFilesCommand zcat \
--quantMode GeneCounts

2) the gene counts output will be called temp.ReadsPerGene.out.tab, and will have four columns, one with gene names, and three with counts:

column 2:  counts for unstranded RNA-seq
column 3:  counts for the 1st read strand aligned with RNA (htseq-count option -s yes)
column 4:  counts for the 2nd read strand aligned with RNA (htseq-count option -s reverse)

You know already your data is stranded, so inspect columns 3 and 4 to decide which one is correct. The correct one will have lots of counts, whilst the other one will have very few counts.

3) if the counts are accumulated at the 3rd column, it means you have to use --SS_lib_type F with Trinity, whereas if the counts are accumulated on the 4rth column, you have to use --SS_lib_type R.

[Moneeb Bajwa]

Ok thank you. I actually found the strandness in the link I provided in the question.

Answer 1

[h.mon]

Sometimes the information is found at each sample page, for example, for the first and last samples (I didn't look at the others):

Construction protocol: Agilent SureSelect Strand Specific RNA

[Moneeb Bajwa]

But I can't find the manual for that; the only one I can find is for paired-end reads

[lieven.sterck]

OK, then you know it is strand-specific sequencing, but you still wouldn't know whether it is a forward or a reverse (genomic) strand, no?

Answer 2

[Arindam Ghosh]

If you are looking for STRANDNESS then use infer_experiment.py from RseQC.

Refer http://rseqc.sourceforge.net/ & https://chipster.csc.fi/manual/library-type-summary.html