RNA-seq

[bogdan]

Hi everyone,

on the analysis of the small RNA-seq datasets: after I remove the adaptor/ linker sequences, what alignment programs do you suggest to use (BLAST, BLAT or short read aligners - apparently, I do get some errors with BOWTIE) ? Thanks a lot,


bogdan

[Richard Finney]

What's your goal? To find differential expression? To find alternative splicing? To find novel exons? To find mutations?
How small is "small RNA-seq datasets"?

[bogdan]

Hi Richard,

thanks for your message : the small RNA seq dataset were genrerated following Illumina protocol after selecting RNAs of size 20-60 nt (including miRNAs and piRNAs). We do particularly look for small RNAs (20-40nt) that may derive from repeats, as piRNAs do. At this moment the issue is mappability to custom databases (miRNAs, piRNAs, ALUs, MIRs): namely after adaptor removal, I do align the reads to these custom databases with BOWTIE, but the results are awkward - more specifically, although I do set up a seed minimal length of 20 (-l 20 -n 3), I do notice in the results alignments that are shorter than 16 nt. Is there anything I do miss ? thanks,

bogdan

[Richard Finney]

You have a data set that is "small rna" sequencing, not an "rna-seq" data set that is small. Gotcha. You're probably feeding it less than SEEDLEN length sequences which it obviously handles. You can filter the input to allow only bigger short read sequences. Some bowtie expert may have the magic command line params to do it without the extra up-front work.

[bogdan]

Thanks; well, if someone knows the parameters that avoid the SEEDLEN errors, please let me know.

[natstreet]

You should take a look at microRazerS

I can also recommend the UEA sRNA toolkit as an analysis pipeline (including mapping of reads).

[bogdan]

That is great, thanks so much.

[zee]

Take a look at Novoalign aligner, some special features are available for microRNA

1) 3' adaptor stripping
2) 5' adaptor stripping
3) Hairpin scoring
4) Simple tab-delimited and SAM output
5) Multithreaded for parallelization

We also have a dedicated documentation page for getting started with miRNA analysis.

[bogdan]

A good suggestion, thank you.