Nimblegen linkers

[suludana]

Hi,
has anyone had any experience with sequence capture from Nimblegen?
If I am not wrong, when you capture your sequence you end up with DNA fragments of around 400bases which have linkers 20bases-long at each side, and after DNase treatment, some of the 100b-fragments will still have these linkers. I think it is not possible to get rid of these when running Solexa machine, but is it possible in the analysis step? How do you get rid of, or handle, the linkers in the analysis?


Cheers

[James W. MacDonald]

You can use Bioconductor tools to find and eliminate the linkers. See the example here:

http://bioconductor.org/packages/2.3/bioc/vignettes/Biostrings/inst/doc/Alignments.pdf

The adapter removal part starts on p. 12, showing lots of different ways to do things, and arguably the best method, which allows for substitutions and indels is on p. 17.

Having some experience with R would be a plus, however the code is there so you can just parrot what Patrick does until you get up to speed.

Jim

[yasutake]

Primer

Hello,

How about using the complementary sequence primer to the linker sequence ?

Yasutake

[suludana]

As far as I know Nimblegen does not make protocol adjustments and their linkers are not the Illumina adapters.

[suludana]

Thanks Jim

[SeqCap_RNG]

Hello,

My name is Luke Dannenberg and I am the Global Product Manager, Sequence Capture for Roche NimbleGen. This discussion thread is a good one and if you would to discuss this particular issue or other issues off-line you can email me at:

[email protected]

Hope to hear from you.

Regards,

Luke

[csquared]

Roche/Nimblegen has released a protocol to use the NimbleGen capture arrays with the Illumina sequencer. It involves ligating the amplified material from the array into longer fragments and then starting the Illumina library prep. Other than a few extra steps, the only downside is the Nimblegen adaptors will represent about 20% of your reads so you need to adjust coverage accordingly.

Perhaps Luke will post that protocol.