pooling BAC library

[nhebraeus]

I am looking for strategies to sequence very large BAC library. To save time and cost of barcoding each one, I am wondering if anybody had experience with combinatorial pooling. What is the best protocol for that? Is there any software available to deconvolute results?

Thanks a lot for your suggestions.

[gabrieltw]

Instead of combination pooling, we just use custom barcodes to make library for BACs and then pool everything together at the end for sequencing. How large is the BAC library do you plan on sequencing ?

[nhebraeus]

THanks a lot for your reply.

The library covers mouse genome, so it's a huge project. Unfortunately, creating custom barcodes for each BAC and then pooling might not be very efficient in this case.

[gabrieltw]

How many BACs are you planning on sequencing for each run ? We did this on MiSeq and we usually pool 192 BACs in one run. We have 384 barcodes so we rotate them for each run

[nhebraeus]

Library is ~10X of mouse genome and it's in 528 plates. (So even if we use HiSeq, it's a lot of barcoding and it's very time-consuming).

[rnaeye]

What are trying to achieve exactly?

[nhebraeus]

We have a library of ~200,000 BACs (or 10X coverage) for the mouse strain we would like to sequence on HiSeq.

Doing mini-prep and barcoding each BAC is very very time-consuming and expensive.


My question if anybody heard of or have experience with pooling unbarcoded BACs (across raws, columns, etc of plates) and sequencing multiple pools (aka combinatorial pooling) and then deconvoluting reads back to wells.

I would also appreciate any suggestions for pooling and barcoding strategy for sequencing a very large BAC library .

[Olaf Blue]

Not certain if this wll help but Jay Shendure's group performed a high throughput deep sequencing project on a Fosmid library:

http://www.nature.com/nbt/journal/v29/n1/abs/nbt.1740.html

Olaf