Tuesday, March 23, 2010

Nextera™ libraries and sequencing primers

We are often asked about the transposon end sequence inserted during the Nextera™ library prep procedure (see technology overview), and how this sequence relates to those of the sequencing primers.

I’m running an Illumina sequencer and don’t want to read through the 19-bp transposon sequence. Isn’t this a waste of limited Illumina data?
The Nextera Illumina libraries are sequenced with a custom sequencing primer (provided in the kit). The first read is the genomic fragment. There are no wasted reads with the Illumina libraries.

Are you sure your Nextera-Illumina sequencing primers are compatible with the Illumina system?
Yes. The Nextera sequencing primers have been fully validated and are completely compatible with the standard Illumina sequencing primers. We have even sequenced a standard Illumina library and a Nextera library in the same channel of the same flow cell. The primers do not interfere with each other.

Do you read through the 19-bp transposon sequence on the Roche/454 sequencing platform?
Yes. Nextera libraries are sequenced using the standard Roche/454 primers. As a result, the first reads are the QC key, followed by any added bar coding, followed by the 19-bp transposon sequence. The transposon sequence must be filtered/masked prior to mapping and assembly.
Transposon Sequence: 5'-AGATGTGTATAAGAGACAG-3'

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