Capture and 454

[Layla]

Hello all!

I am very new to capture arrays and 454 sequencing and also this forum. I have been using roche gsMapper and gsAssembler to map sequence reads (after a capture array) to a reference genome and also to create de-novo contigs. I know there is a particular region that is deleted, but how on earth does one go about finding this region using the roche software or bioinformatically? I am totally lost!

Thankyou to anyone who can point me in the right direction

Layla

[Tom Bair]

basic approach

We are working on this problem as well, our basic approach is to pull genomic sequence based on the nimblegen bed file (basically what probes/regions they expect to hybridize to) and construct an artificial chromosome based on nimblegen's probes plus a little bit of padding around the ends we then use gsMapper to align and then query the AlignmentInfo.tsv to find the depth and gaps (just grep > to find all the gaps) the trick is finding which gaps are due to lack of coverage and which ones are due to deletions. If you have enough overall coverage the deletion gaps seem to have many reads that end and start at the same location. We also have made a correlation file to correlate the actual place in the genome vs the bp of the artifical chromosome to then make a bed or wiggle file to display in UCSC to assess if it is something to look at in detail. Anyway just some ideas for you.

[bioinfosm]

what coverage would you feel is 'enough'. With the long 454 reads, I guess it can be pretty low, still reliable!

[Tom Bair]

nimblegen says 15X you though you could probably go as low as 7X for homozygous snps

[Tom Bair]

It looks like Roche software 2.0. has some new tools in the gsMapper to accommodate going from exon capture to HCdiffs. Specifically you can enter the .bed file, UCSC gp files and dbsnp data as a part of the mapping. Just had the bioinformatics webinar so have not played with it but it looks promising for this use case. The 2.0 software is for titanium but is backwards compatible so you should be able to do a reanalysis on older runs.

[bioinfosm]

Is the webinar available online? Looks very useful to have. Has the 454 already released 2.0 ?

[Tom Bair]

I don't think it is. Roche is horrible to find any actual information. I will send you a pdf of the slides though.

[ECO]

Tom, please post it if you feel you can!

[Tom Bair]

Here are the slide from the webinar check starting around slide 30 for the seq capture stuff
GSFLX_Software_2.0.00_what_is_new.ppt.pdf

[Tom Bair]

Found the link to allow people to get signed up for the full webinar

http://www.us.roche-applied-science.com/forms/TitaniumSoftwareTraining

[ATGC_man]

Hi all,

Nimblegen seq capture Vs Raindance tech.

Who know something about Raindance Technologies ?

http://www.raindancetechnologies.com/

http://www.raindancetechnologies.com...enrichment.pdf

Tks.

Have a nice day !