Bonjour,
Lionel Frangeul a écrit :
> Hi,
>
> I try to assemble small viral genomes from cell cultures with Illumina reads.
> Sometimes the complete genome of the host is not available and
> I try to assemble all the reads (host + virus) to obtain my contigs.
> In this case I have 2 important problems for all the NGS assemblers :
> 1) The coverage of my viral contigs are much more higher than the hundred
> thousand
> contigs of the host (300X to 8000X compar to 3X-30X for the host).
The default maximum coverage of Ray is 4294967295 so this should not be
a problem.
There is also an option to ignore anything with too much coverage.
-use-maximum-seed-coverage
Ignores any seed with a coverage depth above this threshold.
The default is 4294967295.
This was added recently and is not in the v2.0.0.
> 2) The coverage of the viral genome are also very heterogeneous (don't know
> why).
Maybe biases in library preparation. Did you use Nextera ?
>
> In the example enclosed (see fig) I obtain 4 contigs of my virus with Ray2.0
> assembly (k30 = best results, try k21 and k35).
If you provided 30, Ray used 29 as Ray can not assemble anything with a
even k-mer value.
For k=35, did you recompile with MAXKMERLENGTH=64 ? If you did not, k=31
was used.
> Very good results compare to other assemblers without any suppl options to
> configure.
Thank you !
>
> but I don't know why these contigs are not group in only one contig.
> When I search the location of these contigs by blastn on my viral genomes, I
> see that
> 3 of these contigs overlap on 53 and 87 bases with 100 % identity and there
> are many reads over all the junctions.
>
Interesting, did you use paired reads ?
If so, what kind ? (see RayOutput/LibraryStatistics.txt)
> Do you have an idea of the cause of this fragmentation ?
>
Your figure shows uneven coverage across the genome. The new algorithms
in Ray (called Ray Méta) work locally in the de Bruijn graph to infer
coverage distribution. For what I see, it may be due to the local
coverage distributions being too different for each of the fragment
(contig) in your assembly.
p.s. Très belle figure !
Sébastien Boisvert
> TIA
>
> P.S. : the genome start and end by repeats.
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