The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing.

摘要: Author(s): Binladen, Jonas; Gilbert, M; Bollback, Jonathan; Panitz, Frank; Bendixen, Christian; Nielsen, Rasmus; Willerslev, Eske | Abstract: BACKGROUND: The invention of the Genome Sequence 20 DNA Sequencing System (454 parallel sequencing platform) has enabled rapid and high-volume production sequence data. Until now, however, individual emulsion PCR (emPCR) reactions subsequent runs have been unable to combine template from multiple individuals, as homologous sequences cannot be subsequently assigned their original sources. METHODOLOGY: We use conventional with 5-nucleotide tagged primers generate amplification products specimens, followed by through high-throughput (GS20, Roche/454 Life Sciences). Each is traced back its source 5tag-analysis. CONCLUSIONS: demonstrate that this new approach enables assignment virtually all generated correct once anomalies are accounted for (miss-assignment ratel0.4%). Therefore, method accurate sources in single GS20 run. observe a bias distribution differently dependent on 5 nucleotide tag. In particular, labelled cytosine heavily overrepresented among final sequences, while those thymine strongly underrepresented. A weaker also exists regards sorted second dinucleotide tags. As results based run, general applicability requires confirmation. However, our experiments 5primer tagging useful which power can applied PCR-based assays products. will value broad range research areas, such comparative genomics, complete mitochondrial analyses, population genetics, phylogenetics.