Hi Allessandro,
if they generate scaffolds I assume you have paired-ends?
Scaffolds are fine, .. and probrably Roche software knows best Roche
data.
But how many contigs generate these 39 scaffolds? (From what you
have written I don't assume
that 933 contigs form 39 clusters, or?).
On the other hand I have here a small phytoplasm genome, which
really makes problems
in assembly. IMHO the real problem is the data quality due to a GC
content of ~26% and some
kind of repetitive nature. The reads themselves are quite short,
~200bp (titanium data!).
I'd love to have an extra sanger or solexa coverage ;-)
just somecomments, no soultion,
Sven
2009/12/4 Alessandro Riccombeni <rikkomba@xxxxxxxxx>
HI,
thanks for the help.
Yes, actually I think it was mentioned that we paid half as usual
for this sequencing, so probably it was at a lower coverage at the
origin.
The sequencing service provided a preassembled dataset as well: 39
scaffolds and 933 contigs.
Some info: 13 Mbs, 2.38% are Ns, GC in the sequence is 36%, largest
scaffold is 1.9 Mb and the smallest is 3Kb.
After my first MIRA run with the 454 only (as they shouldn't have
used any non-454 reads) I was quite clueless about which strategy
did they use to get 39 scaffolds where I got 11000 contigs. As I
wrote, they didn't use any custom adaptor, so I don't know what I
should do as preprocessing goes...
On Thu, Dec 3, 2009 at 8:18 PM, Bastien Chevreux <bach@xxxxxxxxxxxx>
wrote:
On Donnerstag 03 Dezember 2009 Alessandro Riccombeni wrote:
> I guess I am using on MIRA in the wrong way.
> My dataset is composed of 512,000 454 Titanium reads, without any
custom
> vector from the sequencing service. I have to assemble a fungal
genome of
> around 13 MBs.
> [...]
Hi Alessandro,
regardless of what sequencing providers or Roche may tell you ... I
don't
recommend coverages below 20x in 454 sequencing. 30x is a reasonably
good
coverage to get in balance between costs and number of contigs.
Your numbers tell me you have *at most* a theoretical coverage of
16, probably
more in the region of 14 even as 'miramem' still uses the numbers
Roche gave
in the early days (475) though, having seen a few Titanium sets now,
I suspect
having 400 bases as mean length is more accurate. [Notre to self:
change that
ASAP in the miramem estimator]
Even worse, the numbers appended show that MIRA estimates the
average coverage
to be more like 8x or 9x at most. Which is somehwat disastrous.
> [...]
> I got around 10500-11000 large contigs, trying normal, draft and
accurate.
> I am adding info from the output at the end of this message.
Something's wrong with your data, at least that's my impression. In
the most
simplest case you underestimated the size of the genome and it's not
13MB, but
more like 26 MB.
Then there may be the possibility of contamination: was it really
just one
organism which was sequenced?
Also, your bug might be highly repetitive, which adds another
possibility for
a large number of contigs.
And last but not least ... it may be a problem of the sequencing kit
used. If
your organism has high GC (>=60%) and the Titanium data was
generated with a
sequencing kit delivered in the first 7 to 8 month of this year,
then you need
to talk to your provider as they need to talk to Roche.
> I also tried doing a hybrid assembly with 4800 paired Sanger
reads, getting
> around 9700 large contigs. I found out that there are vectors for
the
> Forward and Reverse Sanger reads, so this is surely creating
problems.
> Nonetheless, I expected getting a much better result from my 454
reads.
The 5k paired Sanger won't help for such a fragemented assembly as
the 454
data suggests. Even if you'd trim away the sequencing vectors a bit
better:
with 5k paired end you cant't scaffold 10k contigs.
> My question is: is there something I am (blatantly) doing wrong?
What am I
> overlooking?
> It's my first approach to assembling, so please excuse me for being
> annoying.
Did your sequencing provider give you the result of a Newbler
assembly of the
Titanium data and is it equally disastrous? Then there's nothing you
can do
apart getting more sequences or getting the project resequenced if
it turns
out to be a bad sequencing kit.
Regards,
Bastien
PS: oh, and don't be lured into the "we could try to close with
Solexa" trap
should the provider propose it. Accept only if *they* do the
assembly and
deliver you the result free of charge :-)
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