Issues in switching from GAII PE to Mate Pair?

[plattsa]

Hi,

We've been running GAII with 2x108 and a 64nt spacer (280nt insert) without problems for a while. We've also been running long insert mate pair reads on 454 with a few more problems (library over-reading, missing adapter sequences, higher costs etc).

What I'd like to do is to try shifting over to 5kbase insert GAII mate pair runs. But I'm going blind into this in terms of any new sequencing or analysis issues we may face. Could anyone provide first hand advice on whether the mate-pair procedure on GAII runs as smoothly as a normal short insert PE run?

Thanks!

[henry.wood]

Hello

I've tried the mate pair with 3kb inserts and it was quite troublesome. The sequencing worked OK, but there were problems in library prep. We lost large amounts of DNA in the various fragmentation and circularisation steps. Once we got the data, it became apparent that a significant proportion of the reads weren't outward facing mate pair reads, but standard paired end reads. This not only reduced the numbers of useable reads, but also proved quite messy to work out which was which.
It might just be that we were doing something wrong, but we've been doing standard library prep for a while now with no real problems.
Good luck if you decide to have a go.

Henry

[plattsa]

Thanks Henry - that does seem to raise a few warnings.

Adrian