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

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 fr...

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Bibliographic Details
Published in:PloS one 2007-02, Vol.2 (2), p.e197-e197
Main Authors: Binladen, Jonas, Gilbert, M Thomas P, Bollback, Jonathan P, Panitz, Frank, Bendixen, Christian, Nielsen, Rasmus, Willerslev, Eske
Format: Article
Language:English
Subjects:
Amplification
Animals
Base Sequence
Bias
Bioinformatics
Biology
Biotechnology
Campylobacter jejuni
Cloning
Computational Biology/Comparative Sequence Analysis
Computational Biology/Population Genetics
Cytosine
Deoxyribonucleic acid
DNA
DNA Primers
DNA sequencing
DNA, Mitochondrial - genetics
Evolutionary Biology
Evolutionary Biology/Evolutionary and Comparative Genetics
Evolutionary Biology/Paleontology
Fines & penalties
Gene Amplification
Gene sequencing
Genetics
Genomes
High-Throughput Screening Assays - instrumentation
High-Throughput Screening Assays - methods
Homology
Humans
Mitochondria
Molecular Sequence Data
Mutation
Nuclear electric power generation
Nucleotide sequence
Phylogeny
Polymerase chain reaction
Polymerase Chain Reaction - instrumentation
Polymerase Chain Reaction - methods
Population genetics
Primers
Product introduction
Pyrimidines
Sequence Alignment
Sequence Analysis, DNA - methods
Sequence Homology, Nucleic Acid
Species Specificity
Tags
Thymine
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Summary: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. 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. 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
ISSN:1932-6203
1932-6203
DOI:10.1371/journal.pone.0000197