[mira_talk] Re: tweaking Manifest for polyploid genome
- From: Martin Mokrejs <mmokrejs@xxxxxxxxxxxxxxxxxx>
- To: mira_talk@xxxxxxxxxxxxx
- Date: Mon, 24 Feb 2014 15:15:35 +0100
Hi Juan,
how many 454 reads do you have on input? I see max coverage 180830
for the 454 technology. also from "Coverage assessment" I see that
only 454 is covered at 0.78x and Solexa at 28.34x. Let me suspect your
main issue is proper trimming of raw reads. Looks the reads just do not
assemble or you sequenced too few material using 454. Ah, I see in your
Manifest file you used RapidLib approach and MIDs ... poor boy, how
did you remove them?
Maybe you would appreciate as a paid service my help, please see
http://www.bioinformatics.cz . There are plenty of tricks needed to get
454 trimming right and I don't know any other tool (except mine) ;)
doing that right, not even of a tool doing the proper queries for all
adapters, primers, artifacts. However, a lot of effort had to be put
into the wrapper code to manage and interpret the candidate alignments.
Having just the right queries is not enough. 3 years of work, 25k lines
of code in python. My apologies if this this is considered as an Ad,
I couldn't resist.
Martin
Gutierrez, Juan wrote:
> Hi,
>
>
>
> I am trying to do RNA-seq de novo on a polyploid (hexaploid) genome
> using a combination of 454 and illumina 100bp paired ended reads. The
> three copies of each gene are highly similar to one another. I am
> having trouble in separating each one of the three copies into three
> different fully-length assemblies. Most of the times I just get a
> fragment of each of the three copies. I am guessing that when Mira
> finds a difference between highly similar transcripts, it just can’t
> assess if there is a polymorphism between 2 of the copies or a
> sequencing error. In any case, Mira seems to end the assembly way
> before reaching the end of the transcript.
>
>
>
> I have prepared and run 2 different Manifests. I am getting better
> results with Manifest.conf (less number of contigs but longer) than
> with Manifest2.conf (higher number but shorter contigs), so I am
> supposing that I could eventually separate the 3 copies of each gene
> by fine-adjusting the parameters.
>
>
>
> Any suggestion would be greatly appreciated,
>
> Thanks so much!
>
> Juan
>
>
>
>
>
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