Simultaneous digital quantification and fluorescence-based size characterization of massively parallel sequencing libraries.
Due to the high cost of failed runs and suboptimal data yields, quantification and determination of fragment size range are crucial steps in the library preparation process for massively parallel sequencing (or next-generation sequencing). Current library quality control methods commonly involve quantification using real-time quantitative PCR and size determination using gel or capillary electrophoresis. These methods are laborious and subject to a number of significant limitations that can make library calibration unreliable. Herein, we propose and test an alternative method for quality control of sequencing libraries using droplet digital PCR (ddPCR). By exploiting a correlation we have discovered between droplet fluorescence and amplicon size, we achieve the joint quantification and size determination of target DNA with a single ddPCR assay. We demonstrate the accuracy and precision of applying this method to the preparation of sequencing libraries.
Translational Research Program, Public Health Sciences Division, Fred Hutchinson Cancer Research Center, Seattle, WA, USA.
Matthew T
MT
Laurie
Jessica A
JA
Bertout
Sean D
SD
Taylor
Joshua N
JN
Burton
Jay A
JA
Shendure
Jason H
JH
Bielas
D045744:Cell Line, Tumor; D004273:DNA, Neoplasm / Q000032:analysis* / Q000737:chemistry; D015723:Gene Library; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA
D004273:DNA, Neoplasm