[mira_talk] Settings for ddRAD data using EST mode
- From: Magnus Popp <magnus.popp@xxxxxxxxxx>
- To: "mira_talk@xxxxxxxxxxxxx" <mira_talk@xxxxxxxxxxxxx>
- Date: Mon, 6 Oct 2014 16:48:25 +0000
Hi,
I’m attempting to assemble ddRAD data from an iontorrent PGM using the EST mode
of mira 4.0.2. The data comes from genomic DNA that has been completely
digested with two different RE’s, ligated with indexed adaptors, size selected
(inserts ca 300-420bp) and then sequenced. The result is a reduced genome
representation, and in this particular case I end up with 2-3k loci of 350-420
bp for each sample, analogous to ESTs except the contigs aren’t necessarily
coding and don’t have a poly-A tail - so basically not very EST like at all...
What I’ve seen so far using the EST mode is quite promising but I need to get
rid of the 3’ end of the reads in order to minimise the number of IUPAC coded
bases in my consensus.
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.
What I’ve done so far is:
#1
project = casfas
job = est,accurate
readgroup = casfas
data = casfas_18.fastq
technology = iontor
PARAMETERS = COMMON_SETTINGS -AS:nop=5 sep=on IONTOR_SETTINGS -AS:mrl=70 mrpc=5
and then some tests with:
#2
project = casfas
job = est,accurate
readgroup = casfas
data = casfas_18.fastq
technology = iontor
PARAMETERS = COMMON_SETTINGS -AS:nop=5 sep=on IONTOR_SETTINGS -AS:mrl=70 mrpc=5
-CL pec=on cpat=off (and cpat=on too)
and:
#3
project = casfas
job = est,accurate
readgroup = casfas
data = casfas_18.fastq
technology = iontor
PARAMETERS = COMMON_SETTINGS -AS:nop=5 sep=on IONTOR_SETTINGS -AS:mrl=70 mrpc=5
-CL pec=on cpat=off qc=on
While #2 reduce the number of IUPACs with 50% (or even better for some
species), #3 is better than #1 but worse than #2. #3 also gave some odd
increase in number of contigs for my single test run but I have to run a few
more to see if that’s consistent
So, my questions are:
1) Do you have any suggestions on how to reduce the number of IUPACs due to low
quality in the 3’ en of reads?
2) Is there a way of telling mira to clip a contig where it goes below a
certain minimum coverage?
3) And somewhat unrelated - what’s the quality score in the fasta.qual files
and how is it calculated?
Cheers,
Magnus
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