But I can't find the manual for that; the only one I can find is for paired-end reads
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"
2 answers
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
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?
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
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Alignment to the reference is the magic word. If you want a more elaborate answer, please give more details.
I am trying to do a de-novo assembly with Trinity
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)
So would I put F (forward) for when Trinity asks for Ss_lib_type when I am aligning?
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:
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: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 Fwith Trinity, whereas if the counts are accumulated on the 4rth column, you have to use--SS_lib_type R.Ok thank you. I actually found the strandness in the link I provided in the question.