[mira_talk] Re: large hybrid assembly w/ minimal ram

  • From: Robin Kramer <kodream@xxxxxxxxx>
  • To: mira_talk@xxxxxxxxxxxxx
  • Date: Mon, 29 Nov 2010 14:00:03 -0600

Sven,

The problem is that bowtie itself has only limited support for indels
since it isn't a true SW aligner, and Abyss in its scaffolding stage
doesn't support indels(even if bowtie generates them), whatsoever.


I am curious as too your experience with the trans package.  Did it do
a good job?  The last I checked it was something akin to an NxN blast
search and required quite a bit of external configuration to use, and
since it was only a perl script I was guessing that it was itself
quite slow.


On 11/16/10, Wachholtz, Michael <mwachholtz@xxxxxxxxxxx> wrote:
> I recently discovered that Abyss has trans-abyss package. While this
> program is geared towards expression analysis with reference genome,
> it has a merge.pl script. We have have been on the fence about what
> k-mer value to use. The results are hard to interpret, but this
> package will do assembly of all values k-mer from i/2 to i (where i is
> the read length) and merge all the contigs into a final assembly. Our
> computer is quad-core with 25GB RAM. It only takes Abyss less than
> 1hour to assemble ~100,000,000 reads. Very fast!! Since all of our
> illumina reads were filtered to contain mostly 30+ quality scores, we
> just run this assembly through MIRA's fasta2frag program. This will
> output quality score file for the fragments, putting in a value of 30
> for each bp (saves me the work of writing a script for this). Then
> just treat the fragments as sanger reads and do hybrid with our 454
> reads in MIRA. If anyone has done Illumina transcriptome assembly with
> the velvet/oases package instead of abyss, I would like to hear your
> thoughts about the advantages or technique you used. While abyss seems
> to do a fine job of catching SNPs and logging them as "popped
> bubbles", I'm not sure how it handles indels & transcript variants.
> Once we have a complete assembly, our goal is to do RNA-Seq analysis
> with the original Illumina data. While MIRA will catch a large
> majority of SNPs during assembly, some of the SNP/variation data will
> have been lost in the abyss assembly. However this "lost" information
> can easily be found when we map reads using bowtie, bam/sam tools.
>
> On Tue, Nov 16, 2010 at 2:55 PM, Sven Klages
> <sir.svencelot@xxxxxxxxxxxxxx> wrote:
>> oh, yes. I see, .. I just wanted to use it for my own data and was quite
>> astonished ;-)
>> fasta output, no qualities ... not of any use for me neither ..
>> cheers,
>> Sven
>>
>> 2010/11/16 Wachholtz, Michael <mwachholtz@xxxxxxxxxxx>
>>>
>>> I have, but the output is in fasta format with no quality scores. The
>>> only advantage this program has is that it will output how many
>>> identical reads there were. I prefer the fastq program in that it will
>>> retain the quality score of best sequence and will output in fastq
>>> format.
>>>
>>> On Mon, Nov 15, 2010 at 5:18 AM, Sven Klages
>>> <sir.svencelot@xxxxxxxxxxxxxx> wrote:
>>> > Hi Michael,
>>> >
>>> > 2010/11/15 Wachholtz, Michael <mwachholtz@xxxxxxxxxxx>
>>> >>
>>> >> [...]
>>> >>
>>> >> it is safe to use such strict criteria. After that, for each lane, we
>>> >> used the fastq program to collapse/remove any identical reads. This
>>> >
>>> > [...]
>>> >
>>> > just a short question. You have successfuly used the FASTX-Toolkit to
>>> > quality-clip your data;
>>> > this tool collection also contains a program to remove duplicates from
>>> > NGS
>>> > data:
>>> >
>>> > FASTQ/A Collapser
>>> > Collapsing identical sequences in a FASTQ/A file into a single sequence
>>> > (while maintaining reads counts)
>>> >
>>> > Have you tried this for your data?
>>> >
>>> > cheers,
>>> > Sven
>>> >
>>> >
>>>
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>>
>
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