Multiplex pairwise assembly of array-derived DNA oligonucleotides

While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synth...

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Main Authors:	Klein, Jason C., Lajoie, Marc J., Schwartz, Jerrod J., Strauch, Eva-Maria, Nelson, Jorgen, Baker, David, Shendure, Jay
Format:	Online
Language:	English
Published:	Oxford University Press 2016
Online Access:	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797260/

id	pubmed-4797260
recordtype	oai_dc
spelling	pubmed-47972602016-03-21 Multiplex pairwise assembly of array-derived DNA oligonucleotides Klein, Jason C. Lajoie, Marc J. Schwartz, Jerrod J. Strauch, Eva-Maria Nelson, Jorgen Baker, David Shendure, Jay Methods Online While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens. Oxford University Press 2016-03-18 2015-11-08 /pmc/articles/PMC4797260/ /pubmed/26553805 http://dx.doi.org/10.1093/nar/gkv1177 Text en © The Author(s) 2015. Published by Oxford University Press on behalf of Nucleic Acids Research. http://creativecommons.org/licenses/by-nc/4.0/ This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@oup.com
repository_type	Open Access Journal
institution_category	Foreign Institution
institution	US National Center for Biotechnology Information
building	NCBI PubMed
collection	Online Access
language	English
format	Online
author	Klein, Jason C. Lajoie, Marc J. Schwartz, Jerrod J. Strauch, Eva-Maria Nelson, Jorgen Baker, David Shendure, Jay
spellingShingle	Klein, Jason C. Lajoie, Marc J. Schwartz, Jerrod J. Strauch, Eva-Maria Nelson, Jorgen Baker, David Shendure, Jay Multiplex pairwise assembly of array-derived DNA oligonucleotides
author_facet	Klein, Jason C. Lajoie, Marc J. Schwartz, Jerrod J. Strauch, Eva-Maria Nelson, Jorgen Baker, David Shendure, Jay
author_sort	Klein, Jason C.
title	Multiplex pairwise assembly of array-derived DNA oligonucleotides
title_short	Multiplex pairwise assembly of array-derived DNA oligonucleotides
title_full	Multiplex pairwise assembly of array-derived DNA oligonucleotides
title_fullStr	Multiplex pairwise assembly of array-derived DNA oligonucleotides
title_full_unstemmed	Multiplex pairwise assembly of array-derived DNA oligonucleotides
title_sort	multiplex pairwise assembly of array-derived dna oligonucleotides
description	While the cost of DNA sequencing has dropped by five orders of magnitude in the past decade, DNA synthesis remains expensive for many applications. Although DNA microarrays have decreased the cost of oligonucleotide synthesis, the use of array-synthesized oligos in practice is limited by short synthesis lengths, high synthesis error rates, low yield and the challenges of assembling long constructs from complex pools. Toward addressing these issues, we developed a protocol for multiplex pairwise assembly of oligos from array-synthesized oligonucleotide pools. To evaluate the method, we attempted to assemble up to 2271 targets ranging in length from 192–252 bases using pairs of array-synthesized oligos. Within sets of complexity ranging from 131–250 targets, we observed error-free assemblies for 90.5% of all targets. When all 2271 targets were assembled in one reaction, we observed error-free constructs for 70.6%. While the assembly method intrinsically increased accuracy to a small degree, we further increased accuracy by using a high throughput ‘Dial-Out PCR’ protocol, which combines Illumina sequencing with an in-house set of unique PCR tags to selectively amplify perfect assemblies from complex synthetic pools. This approach has broad applicability to DNA assembly and high-throughput functional screens.
publisher	Oxford University Press
publishDate	2016
url	https://www.ncbi.nlm.nih.gov/pmc/articles/PMC4797260/
_version_	1613553836027281408

Multiplex pairwise assembly of array-derived DNA oligonucleotides

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