best protocol for sequencing a gene?

[Vanisha]

Hi guys

What do you think the best way to sequence a 12kb gene in multiple individuals is? Conventional sanger costs are still expensive, advances in high throughput sequencing is more attractive, but which platform?

I have been looking at Illumina TruSeq library prep with GAII sequencing, I would have to multiplex 96 samples on a flowcell, 12 samples per pool, and do single end 36bp reads, allowing 6bp for index barcode.

I have also been looking at the protocol in this paper:

http://www.plosone.org/article/info:doi%2F10.1371%2Fjournal.pone.0017785#s4

which uses position specific barcodes per 96 well plate, pooling and then ligation of a plate specific tag - what are people's thoughts on this? There will be a massive number of PCR reactions as I would need to have approx 27 overlapping fragments of the gene per individual to PCR, followed by addition of the plate specific tag through ligation in library prep step and then sequencing.

thoughts are welcome.

Vanisha

[krobison]

A lot of this is going to depend on the precise structure of your gene (exon lengths & how they are spaced out) and exactly how many individuals you really want to profile. It sounds like you might be talking about 100s of individuals -- are you prepared to make that many libraries (concatamerize, shear, repair, ligate, etc)? Perhaps Nextera works with concatamers, but I haven't seen that in the literature yet (granted, only a couple of Nextera papers are out there) & I'm not sure if it supports that degree of barcoding.

An alternative approach to consider would be to design up to 48 amplicons which can be read as 2x100 with full coverage (i.e. design the amplicon to be no longer than about 150 including the targeting primers, allowing for overlap in the middle to get higher quality there). If you can get access to the Fluidigm AccessArray, then 1 array PCRs 48 samples x 48 primer pairs with minimal work.

[james hadfield]

AccessArray for this works well but you need to design it and get access first.

You might also consider a long-range PCR followed by pooled library prep. Just make the one library and forget about multiplexing. This will give you your variant regions. Then follow up with targeted Sanger sequencing.

Nextera does work on lr_PCRS as well so you might do it this way.

A pooled Illumina straegy of one kind is better than making all the libraries.

How much variation are you expecting. Do you have hotpsots you could sequence first? If the sequence is going to vary in lots of places along your 12kb then making 20 2-D pooled (rows and colums) might be a method to consider.

[Vanisha]

The problem with Sanger sequencing is the cost - I want to resequence in 1000 individuals. Has anyone used the Access Array? How well does it work?

[danielvulrich]

Has anyone used the Access Array? How well does it work?

i haven't tried it myself but some my friends say it is ok




Bad cell phone signal Read. http://www.reema.fr/wikini/newslette...ki=GsmRepeater.


gsm signal booster review. Just read it