Multiple fragment lengths in single 454 titanium run?

[Tom McFarland]

Hi all,

I have two different fragments that I want to sequence with 454 titanium. One is about 400 bp long and the other about 500 bp long (including gene-specific primers but not fusion primers). Can I run these together in a single run? A lab mate told me that a disproportionately high number of reads will come from the shorter fragment, but it seems a lot of people are doing multiple fragment lengths and getting good results. I have about 100 samples of the 400 bp fragment and about 60 samples of the 500 bp fragment and was hoping to get by with a 1/4 plate run.

Thanks for any help.

[Nicole 454 Sequencing]

Hi Tom,

We recommend that amplicons be designed between 200-600 base pairs and within approximately 150 base pairs of each other. Smaller fragments will be preferentially amplified when fragment lengths span outside of these recommendations. With that said, your fragments of 400 bp and 500 bp can be sequenced in a single run.

APP No. 001-2010 gives some guidance on Amplicon Sequencing using the GS FLX Titanium platform. This Application Brief and additional references can be found on our customer-accessible website at www.454.com/my454.

Please don’t hesitate to contact your local Roche representative with further queries.

Kind Regards,

Nicole
Technical Support Scientist
454 Life Sciences, A Roche Company

[Tom McFarland]

Thanks

Thank you for your help.

[vlee2]

When we sequence different sizes of amplicons, we separate them in groups by sizes, emPCR, and then combine DNA beads together for sequencing.