Barcoding strategies for multiplexing of samples using a long-read sequencing technology.

Author(s): Lee, Walter and Bowen, Tyson and Harting, John and Hsu, David and Mollova, Emilia and Lee, Eun and Lam, Regina and Hepler, Lance and Bowman, Brett and Brown, Michael and Pinaula, Simeon and Jiao, Phil and Alexander, David and Miller, Erik

We have developed barcoding reagents and workflows for multiplexing amplicons or fragmented native genomic (DNA) prior to Single Molecule, Real-Time (SMRT) Sequencing. The long reads of PacBio’s SMRT Sequencing enable detection of linked mutations across multiple kilobases (kb) of sequence. This feature is particularly useful in the context of mutational analysis or SNP confirmation, where a large number of samples are generated routinely. To validate this workflow, a set of 384 1.7-kb amplicons, each derived from variants of the Phi29 DNA polymerase gene, were barcoded during amplification, pooled, and sequenced on a single SMRT Cell. To demonstrate the applicability of the method to longer inserts, a library of 96 5-kb clones derived from the E. coli genome was sequenced.

Organization: PacBio
Year: 2015

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