Library prep and 454 Sequencing
updated 11/29/11
How do I create an amplicon library for 454 sequencing?
There are two general methods for preparing an amplicon library for 454 sequencing. Fusion primers can be purchased from IDT and used for Lib-A emPCR and bi-directional sequencing (see Roche’s amplicon guidelines), or the amplicon sample can go right into Rapid Library Preparation and Lib-L emPCR.
Because we introduced a barcode and Primer ID in the cDNA synthesis step, and amplified the tagged templates by nested PCR, we used the Rapid Library Preparation with Lib-L emPCR. To adapt amplicons to this protocol, 500ng of equimolarly pooled sample went into end repair.
What is the difference between Lib-A and Lib-L?
Lib-A emPCR will only be done on samples that have amplicon fusion adaptors. Lib-L emPCR is for a whole genome sample preparation or for amplicon samples that have gone through the Rapid Library Preparation. The two library preparations are not cross compatible.
For each library, 4 small volume emulsions with different library concentrations will be completed to determine the optimal molecules/bead ratio for 8% enrichment. For Lib-A, 8 reactions, or 4 per adaptor, are done. By using two different adaptors/emPCR preparations, bidirectional sequencing can be done. However, the Rapid Library preparation is a blunt ended reaction, therefore the amplicon insert can go in either direction.
© 2011 by C. Jabara