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Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells

Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating...

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Autores principales: Marcy, Yann, Ishoey, Thomas, Lasken, Roger S, Stockwell, Timothy B, Walenz, Brian P, Halpern, Aaron L, Beeson, Karen Y, Goldberg, Susanne M. D, Quake, Stephen R
Formato: Texto
Lenguaje:English
Publicado: Public Library of Science 2007
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1988849/
https://www.ncbi.nlm.nih.gov/pubmed/17892324
http://dx.doi.org/10.1371/journal.pgen.0030155
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author Marcy, Yann
Ishoey, Thomas
Lasken, Roger S
Stockwell, Timothy B
Walenz, Brian P
Halpern, Aaron L
Beeson, Karen Y
Goldberg, Susanne M. D
Quake, Stephen R
author_facet Marcy, Yann
Ishoey, Thomas
Lasken, Roger S
Stockwell, Timothy B
Walenz, Brian P
Halpern, Aaron L
Beeson, Karen Y
Goldberg, Susanne M. D
Quake, Stephen R
author_sort Marcy, Yann
collection PubMed
description Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells.
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spelling pubmed-19888492007-09-27 Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells Marcy, Yann Ishoey, Thomas Lasken, Roger S Stockwell, Timothy B Walenz, Brian P Halpern, Aaron L Beeson, Karen Y Goldberg, Susanne M. D Quake, Stephen R PLoS Genet Research Article Since only a small fraction of environmental bacteria are amenable to laboratory culture, there is great interest in genomic sequencing directly from single cells. Sufficient DNA for sequencing can be obtained from one cell by the Multiple Displacement Amplification (MDA) method, thereby eliminating the need to develop culture methods. Here we used a microfluidic device to isolate individual Escherichia coli and amplify genomic DNA by MDA in 60-nl reactions. Our results confirm a report that reduced MDA reaction volume lowers nonspecific synthesis that can result from contaminant DNA templates and unfavourable interaction between primers. The quality of the genome amplification was assessed by qPCR and compared favourably to single-cell amplifications performed in standard 50-μl volumes. Amplification bias was greatly reduced in nanoliter volumes, thereby providing a more even representation of all sequences. Single-cell amplicons from both microliter and nanoliter volumes provided high-quality sequence data by high-throughput pyrosequencing, thereby demonstrating a straightforward route to sequencing genomes from single cells. Public Library of Science 2007-09 2007-09-21 /pmc/articles/PMC1988849/ /pubmed/17892324 http://dx.doi.org/10.1371/journal.pgen.0030155 Text en Copyright: © 2007 Marcy et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
spellingShingle Research Article
Marcy, Yann
Ishoey, Thomas
Lasken, Roger S
Stockwell, Timothy B
Walenz, Brian P
Halpern, Aaron L
Beeson, Karen Y
Goldberg, Susanne M. D
Quake, Stephen R
Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells
title Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells
title_full Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells
title_fullStr Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells
title_full_unstemmed Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells
title_short Nanoliter Reactors Improve Multiple Displacement Amplification of Genomes from Single Cells
title_sort nanoliter reactors improve multiple displacement amplification of genomes from single cells
topic Research Article
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC1988849/
https://www.ncbi.nlm.nih.gov/pubmed/17892324
http://dx.doi.org/10.1371/journal.pgen.0030155
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