[mira_talk] Re: MIRA run failing due to megahubs
- From: Bastien Chevreux <bach@xxxxxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Wed, 25 Jun 2014 20:57:06 +0200
On 25 Jun 2014, at 17:36 , Offord, Victoria <vofford@xxxxxxxxx> wrote:
> […]
> Does anyone have any advice on how I can avoid this? I have copied in the
> manifest below.
Phew … this thread is one reason I like the people on this list. There can be
weeks of calm, but a seemingly simple question can lead to very interesting
threads.
OK, there were already pretty good answers on the list already and here’s just
a couple of my thoughts to recapitulate what was already said and add a couple
of new thigs:
1. most important: get the rRNA out. If you already know the sequence:
mirabait. If you don’t: try sortmerna (never used it) or reconstruct yourself
with small test assemblies (100k to 200k, look for highest expressed
transcripts, then use mirabait with confirmed (BLAST at NCBI) sequences). If
you find good hits at NCBI that also contain ITS (internal transcribed
spacers), also filter these sequences completely away (ITS can also reach very
high expression)
2. When using Illumina, don’t do quality clip before going into MIRA as it does
a better job than simpler clippers that just look at quality.
3. With Illumina data, you can do an adapter removal prior to MIRA if you want.
I don’t think it is necessary, but hey :-)
4. Never run two poly-A removers on a data-set. If you did a poly-A removal
prior to MIRA, you should switch off the one in MIRA as this may then remove
legitimate poly-A/T CDS sequences which have a fringe similarity to poly-A tails
5. The number of megahubs in your data set is pretty small, allowing it to
continue via -SK:mmhr will not harm
6. After the above (especially the rRNA in point 1), maybe you want to relax
the -HS:nrr setting: you’ve used 15, which is not really necessary.
B.
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