PacBio sequencing output increased through uniform and directional fivefold concatenation

Abstract Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer large sequences. Methods for sequencing mid-sized sequences of 600–5,000 bp are currently l...

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Autores principales:	Nisha Kanwar, Celia Blanco, Irene A. Chen, Burckhard Seelig
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/e15146f13e9d454ea27128ae5e18d288
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spelling	oai:doaj.org-article:e15146f13e9d454ea27128ae5e18d2882021-12-02T17:19:14ZPacBio sequencing output increased through uniform and directional fivefold concatenation10.1038/s41598-021-96829-z2045-2322https://doaj.org/article/e15146f13e9d454ea27128ae5e18d2882021-09-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-96829-zhttps://doaj.org/toc/2045-2322Abstract Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer large sequences. Methods for sequencing mid-sized sequences of 600–5,000 bp are currently less efficient. For example, the PacBio Sequel I system yields ~ 100,000–300,000 reads with an accuracy per base pair of 90–99%. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost.Nisha KanwarCelia BlancoIrene A. ChenBurckhard SeeligNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-13 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Nisha Kanwar Celia Blanco Irene A. Chen Burckhard Seelig PacBio sequencing output increased through uniform and directional fivefold concatenation
description	Abstract Advances in sequencing technology have allowed researchers to sequence DNA with greater ease and at decreasing costs. Main developments have focused on either sequencing many short sequences or fewer large sequences. Methods for sequencing mid-sized sequences of 600–5,000 bp are currently less efficient. For example, the PacBio Sequel I system yields ~ 100,000–300,000 reads with an accuracy per base pair of 90–99%. We sought to sequence several DNA populations of ~ 870 bp in length with a sequencing accuracy of 99% and to the greatest depth possible. We optimised a simple, robust method to concatenate genes of ~ 870 bp five times and then sequenced the resulting DNA of ~ 5,000 bp by PacBioSMRT long-read sequencing. Our method improved upon previously published concatenation attempts, leading to a greater sequencing depth, high-quality reads and limited sample preparation at little expense. We applied this efficient concatenation protocol to sequence nine DNA populations from a protein engineering study. The improved method is accompanied by a simple and user-friendly analysis pipeline, DeCatCounter, to sequence medium-length sequences efficiently at one-fifth of the cost.
format	article
author	Nisha Kanwar Celia Blanco Irene A. Chen Burckhard Seelig
author_facet	Nisha Kanwar Celia Blanco Irene A. Chen Burckhard Seelig
author_sort	Nisha Kanwar
title	PacBio sequencing output increased through uniform and directional fivefold concatenation
title_short	PacBio sequencing output increased through uniform and directional fivefold concatenation
title_full	PacBio sequencing output increased through uniform and directional fivefold concatenation
title_fullStr	PacBio sequencing output increased through uniform and directional fivefold concatenation
title_full_unstemmed	PacBio sequencing output increased through uniform and directional fivefold concatenation
title_sort	pacbio sequencing output increased through uniform and directional fivefold concatenation
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/e15146f13e9d454ea27128ae5e18d288
work_keys_str_mv	AT nishakanwar pacbiosequencingoutputincreasedthroughuniformanddirectionalfivefoldconcatenation AT celiablanco pacbiosequencingoutputincreasedthroughuniformanddirectionalfivefoldconcatenation AT ireneachen pacbiosequencingoutputincreasedthroughuniformanddirectionalfivefoldconcatenation AT burckhardseelig pacbiosequencingoutputincreasedthroughuniformanddirectionalfivefoldconcatenation
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PacBio sequencing output increased through uniform and directional fivefold concatenation

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