title: Multiplex picoliter-droplet digital PCR for quantitative assessment of DNA integrity in clinical samples.
abstract: BACKGROUND
  Assessment of DNA integrity and quantity remains a bottleneck for high-throughput molecular genotyping technologies, including next-generation sequencing. In particular, DNA extracted from paraffin-embedded tissues, a major potential source of tumor DNA, varies widely in quality, leading to unpredictable sequencing data. We describe a picoliter droplet-based digital PCR method that enables simultaneous detection of DNA integrity and the quantity of amplifiable DNA.
  
  
  METHODS
  Using a multiplex assay, we detected 4 different target lengths (78, 159, 197, and 550 bp). Assays were validated with human genomic DNA fragmented to sizes of 170 bp to 3000 bp. The technique was validated with DNA quantities as low as 1 ng. We evaluated 12 DNA samples extracted from paraffin-embedded lung adenocarcinoma tissues.
  
  
  RESULTS
  One sample contained no amplifiable DNA. The fractions of amplifiable DNA for the 11 other samples were between 0.05% and 10.1% for 78-bp fragments and ≤1% for longer fragments. Four samples were chosen for enrichment and next-generation sequencing. The quality of the sequencing data was in agreement with the results of the DNA-integrity test. Specifically, DNA with low integrity yielded sequencing results with lower levels of coverage and uniformity and had higher levels of false-positive variants.
  
  
  CONCLUSIONS
  The development of DNA-quality assays will enable researchers to downselect samples or process more DNA to achieve reliable genome sequencing with the highest possible efficiency of cost and effort, as well as minimize the waste of precious samples.
authors: Université Paris Sorbonne Cité, INSERM UMR-S775, Paris, France.
authors: Audrey
authors: A
authors: Didelot
authors: Steve K
authors: SK
authors: Kotsopoulos
authors: Audrey
authors: A
authors: Lupo
authors: Deniz
authors: D
authors: Pekin
authors: Xinyu
authors: X
authors: Li
authors: Ivan
authors: I
authors: Atochin
authors: Preethi
authors: P
authors: Srinivasan
authors: Qun
authors: Q
authors: Zhong
authors: Jeff
authors: J
authors: Olson
authors: Darren R
authors: DR
authors: Link
authors: Pierre
authors: P
authors: Laurent-Puig
authors: Hélène
authors: H
authors: Blons
authors: J Brian
authors: JB
authors: Hutchison
authors: Valerie
authors: V
authors: Taly
mesh_terms: D053938:DNA Fragmentation; D004273:DNA, Neoplasm / Q000032:analysis*; D015894:Genome, Human; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D006801:Humans; D008175:Lung Neoplasms / Q000737:chemistry; D060885:Multiplex Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*
chemical_list: D004273:DNA, Neoplasm