[mira_talk] Re: Settings for ddRAD data using EST mode
- From: Magnus Popp <magnus.popp@xxxxxxxxxx>
- To: "mira_talk@xxxxxxxxxxxxx" <mira_talk@xxxxxxxxxxxxx>
- Date: Wed, 8 Oct 2014 16:01:20 +0000
Hi and thanks for the reply!
Concerning your examples below, I’m OK with the first case as I would interpret
an IUPAC "R" as a result of merging what probably are two alleles, but ideally
mira would give me two contigs. I see that wasn’t what I wrote in my question,
but that’s what I meant, sorry…
In second example the preferred result would be that the contig (or perhaps the
read) is clipped when the quality drops that low.
I will align each RAD loci/“EST” across a handfull of fairly closely related
species and although the errors (be it a single base or a IUPAC base) most of
the time only will result in apomorphic characters, they will mess up the
diversity and branch length estimates in my phylogenetic analyses. So I rather
have somewhat shorter RAD loci/ESTs with ± reliable data than longer ones with
shaky 3’ ends at this stage.
Unless you suggest otherwise, I’ll probably use miraconvert and set -q to
something that works with my data and then simply truncate at the first N or
perhaps just merge the fasta and qual files and use a suitable cut off in fastq
quality trimmer.
Thanks again!
Cheers,
MAgnus
On 8 Oct 2014, at 13:41, Bastien Chevreux
<bach@xxxxxxxxxxxx<mailto:bach@xxxxxxxxxxxx>> wrote:
On October 6, 2014 at 6:48 PM Magnus Popp
<magnus.popp@xxxxxxxxxx<mailto:magnus.popp@xxxxxxxxxx>> wrote:
To complicate matters, there's some heterozygosity in my organisms (di- and
possibly tetraploid plants)
so I only really want to get rid of IUPAC calls stemming from poor quality,
not completely eliminate them.
As this is iontorrent PGM data, the read length varies quite a bit and many
contigs have a 3’ tail consisting of very few (1-4) reads.
MIRA usually tries to get you unambiguous calls and only falls back to IUPAC if
that fails. From what you wrote you would hope for MIRA to give you
- e.g.: an IUPAC in case it needs to decide between an A at qual 60 at a G at
qual 61
- e.g.: a single base G in case it needs to decide between an A at qual 6 at a
G at qual 7
Am I summarising correctly? If yes: can you tell me why you think that this is a
good idea? I do have some trouble at grasping the reasoning for this.
1) Do you have any suggestions on how to reduce the number of IUPACs due to
low quality in the 3’ en of reads?
Clip the 3' a bit harder? Not ideal, I know.
2) Is there a way of telling mira to clip a contig where it goes below a
certain minimum coverage?
No. I think there was a functionality in miraconvert which would N out consensus
on the coverage being below a given level, but atm I see that only wrt to
quality (see -q parameter). I'm not sure why I dropped the version with coverage
... need to check.
3) And somewhat unrelated - what’s the quality score in the fasta.qual files
and how is it calculated?
//www.freelists.org/post/mira_talk/Quality-Values,4
HTH,
B.
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__________________________________________
Magnus Popp
Natural History Museum
University of Oslo
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NO-0318 Oslo, Norway
Phone: +47 22851875
Fax: +47 22851835
Visiting address: Office 112C, Botanical Museum, Sars gate 1, Tøyen
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www.forbio.uio.no/
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