BAC sequencing with Illumina

[JuanLugo]

Hello everyone!

I am new to this sequencing method so I need help. I am planning to sequence a BAC library from a specific genomic region of two different species with Illumina. This region has many repetitive sequences. So, what plan I can adopt to sequence this region. Also, I want to know if there is any oublication using this strategy, because all that I had found is whole genome sequencing or using 454.

Hope you can help me to solve this.

Greetings,
Juan.

[james hadfield]

Long paired end reads will help anchor reads where one end is in a repetetive region. You wil get huge coverage from an Ilumina run so it might be worth sequencing serveral BACs at the same time. Possibly tiling the region your interested in?

[tonybolger]

Hello everyone!

I am new to this sequencing method so I need help. I am planning to sequence a BAC library from a specific genomic region of two different species with Illumina. This region has many repetitive sequences. So, what plan I can adopt to sequence this region. Also, I want to know if there is any oublication using this strategy, because all that I had found is whole genome sequencing or using 454.

The main question is whether you have a usable reference (from a closely related species), and just how big, common and similar those (near-)repeats are. Long repeats, with lots of similar or identical copies means pain.

There is no essential difference between assembling a whole genome and a BAC, except the BAC is smaller, so achieving good coverage is easier, and the data set smaller. The need for paired data spanning the repeats and anchoring into unique sequence is the same in BAC and whole genome, and will be the limiting factor.

How 'good' do you want/need the result to be? There is a chance that a single lane of illumina (with barcoding) would do, but it depends on the answers to the above.