Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Joanna M. Łopacińska-Jørgensen; Jonas N. Pedersen; Mads Bak; Mana M. Mehrjouy; Kristian T. Sørensen; Peter F. Østergaard; Brian Bilenberg; Anders Kristensen; Rafael J. Taboryski; Henrik Flyvbjerg; Rodolphe Marie; Niels Tommerup; Asli Silahtaroglu

doi:10.1038/s41598-017-18091-6

Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

Joanna M. Łopacińska-Jørgensen, Jonas N. Pedersen, Mads Bak, Mana M. Mehrjouy, Kristian T. Sørensen, Peter F. Østergaard, Brian Bilenberg, Anders Kristensen, Rafael J. Taboryski, Henrik Flyvbjerg, Rodolphe Marie, Niels Tommerup, Asli Silahtaroglu^*

^*Corresponding author for this work

Medical Genetics Program

2 Citations (Scopus)

57 Downloads (Pure)

Abstract

Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

Original language	English
Article number	17893
Journal	Scientific Reports
Volume	7
Issue number	1
Number of pages	10
ISSN	2045-2322
DOIs	https://doi.org/10.1038/s41598-017-18091-6
Publication status	Published - Dec 2017

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Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencingFinal published version, 2.37 MBLicence: CC BY

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Łopacińska-Jørgensen, J. M., Pedersen, J. N., Bak, M., Mehrjouy, M. M., Sørensen, K. T., Østergaard, P. F., Bilenberg, B., Kristensen, A., Taboryski, R. J., Flyvbjerg, H., Marie, R., Tommerup, N., & Silahtaroglu, A. (2017). Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing. Scientific Reports, 7(1), Article 17893. https://doi.org/10.1038/s41598-017-18091-6

Łopacińska-Jørgensen, JM, Pedersen, JN, Bak, M, Mehrjouy, MM, Sørensen, KT, Østergaard, PF, Bilenberg, B, Kristensen, A, Taboryski, RJ, Flyvbjerg, H, Marie, R, Tommerup, N & Silahtaroglu, A 2017, 'Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing', Scientific Reports, vol. 7, no. 1, 17893. https://doi.org/10.1038/s41598-017-18091-6

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title = "Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing",

abstract = "Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.",

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doi = "10.1038/s41598-017-18091-6",

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AU - Łopacińska-Jørgensen, Joanna M.

AU - Pedersen, Jonas N.

AU - Bak, Mads

AU - Mehrjouy, Mana M.

AU - Sørensen, Kristian T.

AU - Østergaard, Peter F.

AU - Bilenberg, Brian

AU - Kristensen, Anders

AU - Taboryski, Rafael J.

AU - Flyvbjerg, Henrik

AU - Marie, Rodolphe

AU - Tommerup, Niels

AU - Silahtaroglu, Asli

PY - 2017/12

Y1 - 2017/12

N2 - Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

AB - Next-generation sequencing (NGS) has caused a revolution, yet left a gap: long-range genetic information from native, non-amplified DNA fragments is unavailable. It might be obtained by optical mapping of megabase-sized DNA molecules. Frequently only a specific genomic region is of interest, so here we introduce a method for selection and enrichment of megabase-sized DNA molecules intended for single-molecule optical mapping: DNA from a human cell line is digested by the NotI rare-cutting enzyme and size-selected by pulsed-field gel electrophoresis. For demonstration, more than 600 sub-megabase- to megabase-sized DNA molecules were recovered from the gel and analysed by denaturation-renaturation optical mapping. Size-selected molecules from the same gel were sequenced by NGS. The optically mapped molecules and the NGS reads showed enrichment from regions defined by NotI restriction sites. We demonstrate that the unannotated genome can be characterized in a locus-specific manner via molecules partially overlapping with the annotated genome. The method is a promising tool for investigation of structural variants in enriched human genomic regions for both research and diagnostic purposes. Our enrichment method could potentially work with other genomes or target specified regions by applying other genomic editing tools, such as the CRISPR/Cas9 system.

U2 - 10.1038/s41598-017-18091-6

DO - 10.1038/s41598-017-18091-6

M3 - Journal article

C2 - 29263336

AN - SCOPUS:85038878335

SN - 2045-2322

VL - 7

JO - Scientific Reports

JF - Scientific Reports

IS - 1

M1 - 17893

ER -

Enrichment of megabase-sized DNA molecules for single-molecule optical mapping and next-generation sequencing

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