[mira_talk] Re: prescreening illumina sequences
- From: "Reith, Michael" <Michael.Reith@xxxxxxxxxxxxxx>
- To: <mira_talk@xxxxxxxxxxxxx>
- Date: Tue, 1 Dec 2009 10:49:33 -0500
Thanks for the comments Bastien. It turns out that I have 36% N's (from
paired end, 76 bp reads from a lower eukaryote (genome ~35 Mb)). Two
questions:
1. Since filling up the computer memory with a ton of N's seems like a
waste, I'm going to remove all sequences that don't have at least 10
bases of Phred > 15. If I also chop off the runs of N's at the 3' end
the remaining sequences, is Mira OK with having different length Solexa
sequences, or should I just let Mira do the chopping? I'm a bit worried
about bumping up against the memory limit of our machine (32 Gb).
2. Should I be complaining to my sequencing service? Has anyone had
experience with these read lengths from paired ends? Is this typical or
indicative of problems with the sequencing and/or base calling?
Thanks for your help.
Mike
-----Original Message-----
From: mira_talk-bounce@xxxxxxxxxxxxx
[mailto:mira_talk-bounce@xxxxxxxxxxxxx] On Behalf Of Bastien Chevreux
Sent: November 30, 2009 2:24 PM
To: mira_talk@xxxxxxxxxxxxx
Subject: [mira_talk] Re: prescreening illumina sequences
On Sonntag 29 November 2009 Reith, Michael wrote:
> Just a quick question about prescreening my Illumina sequences. From
a
> quick look, it appears that my sequencing service has sent
everything,
> including sequences that are entirely N's. It's obvious that these
should
> be removed before starting the Mira assembly, as well as those with
only a
> few good bases. My question is how far do I go - do I keep sequences
that
> have 10 or 15 good bases or should I get rid of everything with more
a few
> N's? This is for 72 bp paired reads.
Hello Michael,
if you don't have too many Ns (like, >=20% of your total bases), then
you
don't need to bother with clipping them (be it by quality or by
character)
before going into assembly. The proposed-end-clip (-CL:pec) takes care
of that
and will make sure that only nice sequences are used. It's extremely
effective
for high-throughput data like Solexa or 454.
The only reason one would like to clip N's before loading is to reduce
memory
imprint of the sequences. But as I wrote, I wouldn't bother for less
than 20%
of total seq (and I've never seen Solexa data set with more than a
percent or
two of N-sequences).
Regards,
Bastien
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