Binladen, Jonas; Gilbert, M Thomas; Bollback, Jonathan PISTA ; Panitz, Frank; Bendixen, Christian; Nielsen, Rasmus; Willerslev, Eske
BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled the rapid and high-volume production of sequence data. Until now, however, individual emulsion PCR (emPCR) reactions and subsequent sequencing runs have been unable to combine template DNA from multiple individuals, as homologous sequences cannot be subsequently assigned to their original sources. METHODOLOGY: We use conventional PCR with 5'-nucleotide tagged primers to generate homologous DNA amplification products from multiple specimens, followed by sequencing through the high-throughput Genome Sequence 20 DNA Sequencing System (GS20, Roche/454 Life Sciences). Each DNA sequence is subsequently traced back to its individual source through 5'tag-analysis. CONCLUSIONS: We demonstrate that this new approach enables the assignment of virtually all the generated DNA sequences to the correct source once sequencing anomalies are accounted for (miss-assignment rate&lt;0.4%). Therefore, the method enables accurate sequencing and assignment of homologous DNA sequences from multiple sources in single high-throughput GS20 run. We observe a bias in the distribution of the differently tagged primers that is dependent on the 5' nucleotide of the tag. In particular, primers 5' labelled with a cytosine are heavily overrepresented among the final sequences, while those 5' labelled with a thymine are strongly underrepresented. A weaker bias also exists with regards to the distribution of the sequences as sorted by the second nucleotide of the dinucleotide tags. As the results are based on a single GS20 run, the general applicability of the approach requires confirmation. However, our experiments demonstrate that 5'primer tagging is a useful method in which the sequencing power of the GS20 can be applied to PCR-based assays of multiple homologous PCR products. The new approach will be of value to a broad range of research areas, such as those of comparative genomics, complete mitochondrial analyses, population genetics, and phylogenetics.
JB and EW were supported by the Wellcome Trust, UK, the Carlsberg Foundation, DK, and the National Science Foundation, DK. MTPG acknowledges the Marie Curie Actions FP6-MEIF-CT-2005-025002 ‘FORMAPLEX’ grant for funding his research. JPB and RN were funded by the Danish FSS and the National Science Foundation, DK. None of the sponsors or funders have had any influence on the data or manuscript presented here.
Binladen J, Gilbert MT, Bollback JP, et al. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS One. 2007;2(2). doi:10.1371/journal.pone.0000197
Binladen, J., Gilbert, M. T., Bollback, J. P., Panitz, F., Bendixen, C., Nielsen, R., & Willerslev, E. (2007). The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS One. Public Library of Science. https://doi.org/10.1371/journal.pone.0000197
Binladen, Jonas, M Thomas Gilbert, Jonathan P Bollback, Frank Panitz, Christian Bendixen, Rasmus Nielsen, and Eske Willerslev. “The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing.” PLoS One. Public Library of Science, 2007. https://doi.org/10.1371/journal.pone.0000197.
J. Binladen et al., “The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing,” PLoS One, vol. 2, no. 2. Public Library of Science, 2007.
Binladen J, Gilbert MT, Bollback JP, Panitz F, Bendixen C, Nielsen R, Willerslev E. 2007. The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing. PLoS One. 2(2).
Binladen, Jonas, et al. “The Use of Coded PCR Primers Enables High-Throughput Sequencing of Multiple Homolog Amplification Products by 454 Parallel Sequencing.” PLoS One, vol. 2, no. 2, Public Library of Science, 2007, doi:10.1371/journal.pone.0000197.