Whole Genome Re-sequencing concept

[Marco0208]

Hello Guys

I am new with Next generation sequencing technology and unfamiliar with the concept. As far as i know Whole genome re-sequencing is where the genome of an individual was sequenced using bridge-PCR method (for Illumina) and compared with a known reference. This technique is use to identify SNPs, SVs, InDel (such mutations).

Am i right so far??? any extra piece i am missing ??

Cheers

[westerman]

Correct so far. Missing pieces would be more details for the sequencing part. "... In the case of Illumina HiSeq 2000, preparation of the sequencing library is done by bridge PCR, while the sequencing is done by a technology referred to as cyclic reversible termination. ..." and perhaps more details on how the computational comparison is done, depth of coverage needed, how to detect false positives, etc.

[Marco0208]

Hi westerman

I appreciate your correction, may i ask for more about depth of coverage .... IE, how is it determined ???? Is it used to detect SNP ??? I did cancer research in my MSC and were focused on monoclonal antibodies so never learn much about it

[TonyBrooks]

A simple way to determine average read depth is to do the following equation

How many of my sequenced bases have I uniquely mapped to my target (genome)
divided by
How big is my target (genome)

i.e. how many times, on average, have I sequenced each base.

The reason why you need high levels of coverage is to compensate for error and systematic bias in next-gen sequencing.

Of course, this is an average, so there will be regions of high coverage and regions of low (zero) coverage.

What depth you need depends on your application. For resequencing a "normal" genome you'll probably get away with 40X coverage.
If you know your sample is highly homogeneous (cell line) you may be able to drop that to 20X.
If you are sequencing a tumor sample (mixed genomes) you may want to go to hundreds, if not thousand fold coverage.

[krobison]

BTW, while Illumina & Bridge PCR are one way, that is not an essential part of the definition, and human genome resequencing has been performed on a number of other platforms (454, Ion Torrent, Helicos, SOLiD & Complete Genomics)