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

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

Section of Forensic Genetics

14 Citations (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.

Translated title of the contribution	Kvantificering af massively parallel sequencing-biblioteker - et komparativt studie af otte metoder
Original language	English
Article number	1110
Journal	Scientific Reports
Volume	8
Number of pages	9
ISSN	2045-2322
Publication status	Published - 1 Dec 2018

Access to Document

Quantification of massively parallel sequencing libraries – a comparative study of eight methodsFinal published version, 1.43 MBLicence: CC BY

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

Cite this

@article{a5acfc214f2d4b0f928b62bedc44efea,

title = "Quantification of massively parallel sequencing libraries - a comparative study of eight methods",

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.",

author = "Christian Hussing and Marie-Louise Kampmann and Mogensen, {Helle Smidt} and Claus B{\o}rsting and Niels Morling",

year = "2018",

month = dec,

day = "1",

language = "English",

volume = "8",

journal = "Scientific Reports",

issn = "2045-2322",

publisher = "nature publishing group",

}

TY - JOUR

T1 - Quantification of massively parallel sequencing libraries - a comparative study of eight methods

AU - Hussing, Christian

AU - Kampmann, Marie-Louise

AU - Mogensen, Helle Smidt

AU - Børsting, Claus

AU - Morling, Niels

PY - 2018/12/1

Y1 - 2018/12/1

N2 - 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.

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.

M3 - Journal article

SN - 2045-2322

VL - 8

JO - Scientific Reports

JF - Scientific Reports

M1 - 1110

ER -