Comparison of DNA Quantification Methods for Next Generation Sequencing

Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rar...

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Bibliographic Details
Published in:Scientific reports 2016-04, Vol.6 (1), p.24067-24067, Article 24067
Main Authors: Robin, Jérôme D., Ludlow, Andrew T., LaRanger, Ryan, Wright, Woodring E., Shay, Jerry W.
Format: Article
Language:English
Subjects:
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Cancer
Chromatin
Crystallography, X-Ray
Density measurement
Deoxyribonucleic acid
DNA
DNA - analysis
DNA sequencing
Fluorometry
G-Quadruplexes
Genome, Human
Genomics - methods
Genotype
Genotyping
Heterogeneity
High-Throughput Nucleotide Sequencing - methods
Humanities and Social Sciences
Humans
Hydrogen Bonding
Magnetic Resonance Spectroscopy - methods
Markov Chains
Models, Statistical
Molecular Dynamics Simulation
multidisciplinary
Nucleic Acid Conformation
Optical density
Polymerase chain reaction
Probability
Protein Denaturation
Regulatory sequences
Science
Science (multidisciplinary)
Thrombin - chemistry
Titration
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Summary:Next Generation Sequencing (NGS) is a powerful tool that depends on loading a precise amount of DNA onto a flowcell. NGS strategies have expanded our ability to investigate genomic phenomena by referencing mutations in cancer and diseases through large-scale genotyping, developing methods to map rare chromatin interactions (4C; 5C and Hi-C) and identifying chromatin features associated with regulatory elements (ChIP-seq, Bis-Seq, ChiA-PET). While many methods are available for DNA library quantification, there is no unambiguous gold standard. Most techniques use PCR to amplify DNA libraries to obtain sufficient quantities for optical density measurement. However, increased PCR cycles can distort the library’s heterogeneity and prevent the detection of rare variants. In this analysis, we compared new digital PCR technologies (droplet digital PCR; ddPCR, ddPCR-Tail) with standard methods for the titration of NGS libraries. DdPCR-Tail is comparable to qPCR and fluorometry (QuBit) and allows sensitive quantification by analysis of barcode repartition after sequencing of multiplexed samples. This study provides a direct comparison between quantification methods throughout a complete sequencing experiment and provides the impetus to use ddPCR-based quantification for improvement of NGS quality.
ISSN:2045-2322
2045-2322
DOI:10.1038/srep24067