Quantification of massively parallel sequencing libraries - a comparative study of eight methods

Christian Hussing; Marie-Louise Kampmann; Helle Smidt Mogensen; Claus Børsting; Niels Morling

Quantification of massively parallel sequencing libraries - a comparative study of eight methods

Bidragets oversatte titel: Kvantificering af massively parallel sequencing-biblioteker - et komparativt studie af otte metoder

Christian Hussing, Marie-Louise Kampmann, Helle Smidt Mogensen, Claus Børsting, Niels Morling

Afdeling for Retsgenetik

14 Citationer (Scopus)

35 Downloads (Pure)

Abstract

Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out.

Bidragets oversatte titel	Kvantificering af massively parallel sequencing-biblioteker - et komparativt studie af otte metoder
Originalsprog	Engelsk
Artikelnummer	1110
Tidsskrift	Scientific Reports
Vol/bind	8
Antal sider	9
ISSN	2045-2322
Status	Udgivet - 1 dec. 2018

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Quantification of massively parallel sequencing libraries – a comparative study of eight methodsForlagets udgivne version, 1,43 MBLicens: CC BY

https://www.nature.com/articles/s41598-018-19574-wLicens: CC BY

Citationsformater

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AB - Quantification of massively parallel sequencing libraries is important for acquisition of monoclonal beads or clusters prior to clonal amplification and to avoid large variations in library coverage when multiple samples are included in one sequencing analysis. No gold standard for quantification of libraries exists. We assessed eight methods of quantification of libraries by quantifying 54 amplicon, six capture, and six shotgun fragment libraries. Chemically synthesized double-stranded DNA was also quantified. Light spectrophotometry, i.e. NanoDrop, was found to give the highest concentration estimates followed by Qubit and electrophoresis-based instruments (Bioanalyzer, TapeStation, GX Touch, and Fragment Analyzer), while SYBR Green and TaqMan based qPCR assays gave the lowest estimates. qPCR gave more accurate predictions of sequencing coverage than Qubit and TapeStation did. Costs, time-consumption, workflow simplicity, and ability to quantify multiple samples are discussed. Technical specifications, advantages, and disadvantages of the various methods are pointed out.

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