Analysis of Base-Position Error Rate of Next-Generation Sequencing to Detect Tumor Mutations in Circulating DNA.
Detecting single-nucleotide variations and insertions/deletions in circulating tumor DNA is challenging because of their low allele frequency. The clinical use of circulating tumor DNA to characterize tumor genetic alterations requires new methods based on next-generation sequencing.



We developed a method based on quantification of error rate of each base position [position error rate (PER)]. To identify mutations, a binomial test was used to compare the minor-allele frequency to the measured PER at each base position. This process was validated in control samples and in 373 plasma samples from patients with lung or pancreatic cancer.



Minimal mutated allele frequencies were 0.003 for single-nucleotide variations and 0.001 for insertions/deletions. Independent testing performed by droplet digital PCR (n = 231 plasma samples) showed strong agreement with the base-PER method (κ = 0.90).



Targeted next-generation sequencing analyzed with the base-PER method represents a robust and low cost method to detect circulating tumor DNA in patients with cancer.
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.|Department of Medical Oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique-Hôpitaux de Paris, Paris, France.
Nicolas
N
Pécuchet
MERIT-UMR IRD 216, Paris Sorbonne Cité Université, Paris, France.
Yves
Y
Rozenholc
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.
Eleonora
E
Zonta
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.
Daniel
D
Pietrasz
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.
Audrey
A
Didelot
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.|Department of Medical Oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique-Hôpitaux de Paris, Paris, France.
Pierre
P
Combe
Department of Pathology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique-Hôpitaux de Paris, Paris, France.
Laure
L
Gibault
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.|Department of Gastro-enterology, Hôpital Pitié-Salpêtrière, Assistance Publique-Hôpitaux de Paris, Paris, France.
Jean-Baptiste
JB
Bachet
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.
Valérie
V
Taly
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.|Department of Medical Oncology, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique-Hôpitaux de Paris, Paris, France.
Elizabeth
E
Fabre
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer.|Department of Biochemistry, Pharmacogenetic and Molecular Oncology Unit, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique-Hôpitaux de Paris, Paris, France.
Hélène
H
Blons
INSERM UMR-S1147, CNRS SNC 5014, Paris Sorbonne Cité Université, Paris, France-Equipe labélisée Ligue Contre le cancer; pierre.laurent-puig@parisdescartes.fr.|Department of Biochemistry, Pharmacogenetic and Molecular Oncology Unit, Hôpital Européen Georges Pompidou (HEGP), Assistance Publique-Hôpitaux de Paris, Paris, France.
Pierre
P
Laurent-Puig
D000483:Alleles; D004273:DNA, Neoplasm / Q000097:blood* / Q000235:genetics*; D059014:High-Throughput Nucleotide Sequencing; D006801:Humans; D008175:Lung Neoplasms / Q000175:diagnosis / Q000235:genetics*; D016254:Mutagenesis, Insertional; D009154:Mutation; D010190:Pancreatic Neoplasms / Q000175:diagnosis / Q000235:genetics*; D020641:Polymorphism, Single Nucleotide / Q000235:genetics; D017422:Sequence Analysis, DNA; D017384:Sequence Deletion
D004273:DNA, Neoplasm