Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.

Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are current...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Ellen Knierim, Barbara Lucke, Jana Marie Schwarz, Markus Schuelke, Dominik Seelow
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2011
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/6e3d7d130cb44204ae24126e3ab13354
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:6e3d7d130cb44204ae24126e3ab13354
record_format	dspace
spelling	oai:doaj.org-article:6e3d7d130cb44204ae24126e3ab133542021-11-18T07:33:19ZSystematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.1932-620310.1371/journal.pone.0028240https://doaj.org/article/6e3d7d130cb44204ae24126e3ab133542011-01-01T00:00:00Zhttps://www.ncbi.nlm.nih.gov/pmc/articles/pmid/22140562/?tool=EBIhttps://doaj.org/toc/1932-6203Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design.Ellen KnierimBarbara LuckeJana Marie SchwarzMarkus SchuelkeDominik SeelowPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 6, Iss 11, p e28240 (2011)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Ellen Knierim Barbara Lucke Jana Marie Schwarz Markus Schuelke Dominik Seelow Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.
description	Next Generation Sequencing (NGS) technologies are gaining importance in the routine clinical diagnostic setting. It is thus desirable to simplify the workflow for high-throughput diagnostics. Fragmentation of DNA is a crucial step for preparation of template libraries and various methods are currently known. Here we evaluated the performance of nebulization, sonication and random enzymatic digestion of long-range PCR products on the results of NGS. All three methods produced high-quality sequencing libraries for the 454 platform. However, if long-range PCR products of different length were pooled equimolarly, sequence coverage drastically dropped for fragments below 3,000 bp. All three methods performed equally well with regard to overall sequence quality (PHRED) and read length. Enzymatic fragmentation showed highest consistency between three library preparations but performed slightly worse than sonication and nebulization with regard to insertions/deletions in the raw sequence reads. After filtering for homopolymer errors, enzymatic fragmentation performed best if compared to the results of classic Sanger sequencing. As the overall performance of all three methods was equal with only minor differences, a fragmentation method can be chosen solely according to lab facilities, feasibility and experimental design.
format	article
author	Ellen Knierim Barbara Lucke Jana Marie Schwarz Markus Schuelke Dominik Seelow
author_facet	Ellen Knierim Barbara Lucke Jana Marie Schwarz Markus Schuelke Dominik Seelow
author_sort	Ellen Knierim
title	Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.
title_short	Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.
title_full	Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.
title_fullStr	Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.
title_full_unstemmed	Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.
title_sort	systematic comparison of three methods for fragmentation of long-range pcr products for next generation sequencing.
publisher	Public Library of Science (PLoS)
publishDate	2011
url	https://doaj.org/article/6e3d7d130cb44204ae24126e3ab13354
work_keys_str_mv	AT ellenknierim systematiccomparisonofthreemethodsforfragmentationoflongrangepcrproductsfornextgenerationsequencing AT barbaralucke systematiccomparisonofthreemethodsforfragmentationoflongrangepcrproductsfornextgenerationsequencing AT janamarieschwarz systematiccomparisonofthreemethodsforfragmentationoflongrangepcrproductsfornextgenerationsequencing AT markusschuelke systematiccomparisonofthreemethodsforfragmentationoflongrangepcrproductsfornextgenerationsequencing AT dominikseelow systematiccomparisonofthreemethodsforfragmentationoflongrangepcrproductsfornextgenerationsequencing
_version_	1718423291747631104

Systematic comparison of three methods for fragmentation of long-range PCR products for next generation sequencing.

Ejemplares similares