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Parallel tagged sequencing on the 454 platform
Matthias Meyer, Udo Stenzel & Michael Hofreiter
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Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, Deutscher Platz 6, D-04103 Leipzig, Germany. Correspondence should be addressed to M.M. ([email protected]).
Published online 31 January 2008; doi:10.1038/nprot.2007.520
Parallel tagged sequencing (PTS) is a molecular barcoding method designed to adapt the recently developed high-throughput 454 parallel sequencing technology for use with multiple samples. Unlike other barcoding methods, PTS can be applied to any type of double-stranded DNA (dsDNA) sample, including shotgun DNA libraries and pools of PCR products, and requires no amplication or gel purication steps. The method relies on attaching sample-specic barcoding adapters, which include sequence tags and a restriction site, to blunt-end repaired DNA samples by ligation and strand-displacement. After pooling multiple barcoded samples, molecules without sequence tags are effectively excluded from sequencing by dephosphorylation and restriction digestion, and using the tag sequences, the source of each DNA sequence can be traced. This protocol allows for sequencing 300 or more complete mitochondrial genomes on a single 454 GS FLX run, or twenty-ve 6-kb plasmid sequences on only one 16th plate region. Most of the reactions can be performed in a multichannel setup on 96-well reaction plates, allowing for processing up to several hundreds of samples in a few days.
INTRODUCTIONRationaleOver the last three decades, Sanger sequencing1 has been the dominant DNA sequencing technology in all areas of life sciences, used to retrieve individual sequences or to decipher entire genomes.Although the throughput of this technology has gradually increased over time, it has now been exceeded by recently developed next-generation sequencing technologies2, such as 454 (Roche)3, Solexa (Illumina)4 and SOLiD (ABI). These technologies have increased the number of sequences obtained in a single run of a machine by several orders of magnitude, from mere hundreds to hundreds of thousands or even millions. Their superior efciency in terms of both cost and time per sequenced nucleotide has not only spawned exploration in new sequencing elds, for example, ultra deep amplicon sequencing5 or paleogenomics6, but has also replaced Sanger sequencing...