Illumina to sequence region of 50 kB

[Marius]

Hi there,

is it possible to somehow entirely sequence a region of 50 kB (reference genome available) from a specific chromosome of one individual with Illumina sequencing (100 cycle runs)? If so, could you give me any references (protocols, studies) describing this method?

Thank you a lot!

[niceday]

overlapping PCR amplicons?
Or you could speak to agilent about producing RNA baits for the 50kb region and do the enrichment.
There will be other ways but these two are just off the top of my head.

[Marius]

Sorry, probably beginner question: by overlapping PCR amplicons you mean just enrich the region of interest with PCR by making several overlapping fragments (how long are those fragments normally?), and then shear them, ligate a Illumina adapter to them and sequence them? - I guess though 454 sequencing would be more appropriate for this, or are 100 cycle Illumina runs long enough to find long enough overlapps between 100bp-fragments to correctly align these afterwards to get the whole 50kB region?

[krobison]

With deep Illumina paired-end coverage you should have no trouble finding the overlaps and assembling such a project. Amplicon length can be quite long, if you use a long PCR kit.

As someone pointed out, Agilent, Nimblegen and others will design custom oligos to cover a region. For a single individual, you'd probably be hybridizing to a microarray. If you don't want to do this yourself, many service providers can handle it.

[Marius]

Thanks a lot for your answers. - How about single-end, would this even be good enough quality? And how about 76 cycles single-end? Since I have some reads left (about 15 Mio) within a 76-single end lane I could use, I wonder if this would even work out (I would barcode the samples). For lets say a 50 kb fragment, how many 76-single end reads would I need to get a coverage high enough to be able to afterwards align these sort sequence to the whole 50 kb fragment?