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Parallel tagged sequencing on the 454 platform

Nature Protocols volume 3, pages 267278 (2008) | Download Citation


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 amplification or gel purification steps. The method relies on attaching sample-specific 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-five 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.

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We thank Christine Green for comments on the manuscript, Ellen Gunnarsdottir for help with providing data and Knut Finstermeier for help with the figures. This work was funded by the Max Planck Society.

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  1. Max Planck Institute for Evolutionary Anthropology, Department of Evolutionary Genetics, Deutscher Platz 6, D-04103 Leipzig, Germany.

    • Matthias Meyer
    • , Udo Stenzel
    •  & Michael Hofreiter


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Correspondence to Matthias Meyer.

Supplementary information

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  1. 1.

    Supplementary Table 1

    Tags for designing barcoding oligos. Listed are 6-, 7- and 8-nt tags with a minimum distance of two or three substitutions. See Fig. 2b for instructions on how to convert the tags into full oligo sequences.

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