title: Comparison of DNA Quantification Methods for Next Generation Sequencing.
abstract: 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.
authors.affiliation: Department of Cell Biology, 5323 Harry Hines Boulevard, UT Southwestern Medical Center, Dallas, TX 75390-9030, USA.
authors.forename: Jérôme D
authors.initials: JD
authors.lastname: Robin
authors.affiliation: Department of Cell Biology, 5323 Harry Hines Boulevard, UT Southwestern Medical Center, Dallas, TX 75390-9030, USA.
authors.forename: Andrew T
authors.initials: AT
authors.lastname: Ludlow
authors.affiliation: Department of Cell Biology, 5323 Harry Hines Boulevard, UT Southwestern Medical Center, Dallas, TX 75390-9030, USA.
authors.forename: Ryan
authors.initials: R
authors.lastname: LaRanger
authors.affiliation: Department of Cell Biology, 5323 Harry Hines Boulevard, UT Southwestern Medical Center, Dallas, TX 75390-9030, USA.
authors.forename: Woodring E
authors.initials: WE
authors.lastname: Wright
authors.affiliation: Department of Cell Biology, 5323 Harry Hines Boulevard, UT Southwestern Medical Center, Dallas, TX 75390-9030, USA.|Center for Excellence in Genomics Medicine Research, King Abdulaziz University, Jeddah, Saudi Arabia.
authors.forename: Jerry W
authors.initials: JW
authors.lastname: Shay
mesh_terms: D018360:Crystallography, X-Ray; D004247:DNA / Q000032:analysis*; D054856:G-Quadruplexes; D015894:Genome, Human; D023281:Genomics / Q000379:methods; D005838:Genotype; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D006860:Hydrogen Bonding; D009682:Magnetic Resonance Spectroscopy / Q000379:methods; D008390:Markov Chains; D015233:Models, Statistical; D056004:Molecular Dynamics Simulation; D009690:Nucleic Acid Conformation; D011336:Probability; D011489:Protein Denaturation; D013917:Thrombin / Q000737:chemistry
chemical_list: D004247:DNA; D013917:Thrombin