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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| Veröffentlicht in: | PloS one 2007-02, Vol.2 (2), p.e197-e197 |
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| creator | Binladen, Jonas Gilbert, M Thomas P Bollback, Jonathan P Panitz, Frank Bendixen, Christian Nielsen, Rasmus Willerslev, Eske |
| description | 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 |
| doi_str_mv | 10.1371/journal.pone.0000197 |
| format | Article |
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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<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.</description><identifier>ISSN: 1932-6203</identifier><identifier>EISSN: 1932-6203</identifier><identifier>DOI: 10.1371/journal.pone.0000197</identifier><identifier>PMID: 17299583</identifier><language>eng</language><publisher>United States: Public Library of Science</publisher><subject>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</subject><ispartof>PloS one, 2007-02, Vol.2 (2), p.e197-e197</ispartof><rights>COPYRIGHT 2007 Public Library of Science</rights><rights>2007 Binladen et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License (the “License”), which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Notwithstanding the ProQuest Terms and Conditions, you may use this content in accordance with the terms of the License.</rights><rights>Binladen et al. 2007</rights><lds50>peer_reviewed</lds50><oa>free_for_read</oa><woscitedreferencessubscribed>false</woscitedreferencessubscribed><citedby>FETCH-LOGICAL-c780t-d1df1d5afcbd1fd3778bc80c9c094786bec7061300c096eebb5147ab5dfca0163</citedby><cites>FETCH-LOGICAL-c780t-d1df1d5afcbd1fd3778bc80c9c094786bec7061300c096eebb5147ab5dfca0163</cites></display><links><openurl>$$Topenurl_article</openurl><openurlfulltext>$$Topenurlfull_article</openurlfulltext><thumbnail>$$Tsyndetics_thumb_exl</thumbnail><linktopdf>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797623/pdf/$$EPDF$$P50$$Gpubmedcentral$$Hfree_for_read</linktopdf><linktohtml>$$Uhttps://www.ncbi.nlm.nih.gov/pmc/articles/PMC1797623/$$EHTML$$P50$$Gpubmedcentral$$Hfree_for_read</linktohtml><link.rule.ids>230,314,723,776,780,860,881,2096,2915,23845,27901,27902,53766,53768,79343,79344</link.rule.ids><backlink>$$Uhttps://www.ncbi.nlm.nih.gov/pubmed/17299583$$D View this record in MEDLINE/PubMed$$Hfree_for_read</backlink></links><search><contributor>Hahn, Matthew</contributor><creatorcontrib>Binladen, Jonas</creatorcontrib><creatorcontrib>Gilbert, M Thomas P</creatorcontrib><creatorcontrib>Bollback, Jonathan P</creatorcontrib><creatorcontrib>Panitz, Frank</creatorcontrib><creatorcontrib>Bendixen, Christian</creatorcontrib><creatorcontrib>Nielsen, Rasmus</creatorcontrib><creatorcontrib>Willerslev, Eske</creatorcontrib><title>The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing</title><title>PloS one</title><addtitle>PLoS One</addtitle><description>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<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.</description><subject>Amplification</subject><subject>Animals</subject><subject>Base Sequence</subject><subject>Bias</subject><subject>Bioinformatics</subject><subject>Biology</subject><subject>Biotechnology</subject><subject>Campylobacter jejuni</subject><subject>Cloning</subject><subject>Computational Biology/Comparative Sequence Analysis</subject><subject>Computational Biology/Population Genetics</subject><subject>Cytosine</subject><subject>Deoxyribonucleic acid</subject><subject>DNA</subject><subject>DNA Primers</subject><subject>DNA sequencing</subject><subject>DNA, Mitochondrial - genetics</subject><subject>Evolutionary Biology</subject><subject>Evolutionary Biology/Evolutionary and Comparative Genetics</subject><subject>Evolutionary Biology/Paleontology</subject><subject>Fines & penalties</subject><subject>Gene Amplification</subject><subject>Gene sequencing</subject><subject>Genetics</subject><subject>Genomes</subject><subject>High-Throughput Screening Assays - 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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<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.</abstract><cop>United States</cop><pub>Public Library of Science</pub><pmid>17299583</pmid><doi>10.1371/journal.pone.0000197</doi><tpages>e197</tpages><oa>free_for_read</oa></addata></record> |
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| identifier | ISSN: 1932-6203 |
| ispartof | PloS one, 2007-02, Vol.2 (2), p.e197-e197 |
| issn | 1932-6203 1932-6203 |
| language | eng |
| recordid | cdi_plos_journals_1950105447 |
| source | MEDLINE; DOAJ Directory of Open Access Journals; Elektronische Zeitschriftenbibliothek - Frei zugängliche E-Journals; PubMed Central; Free Full-Text Journals in Chemistry; Public Library of Science (PLoS) |
| 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 |
| title | The use of coded PCR primers enables high-throughput sequencing of multiple homolog amplification products by 454 parallel sequencing |
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