Mutation detection by clonal sequencing of PCR amplicons and grouped read typing is applicable to clinical diagnostics.
We describe a sensitive technique for mutation detection using clonal sequencing. We analyzed DNA extracted from 13 cancer cell lines and 35 tumor samples and applied a novel approach to identify disease-associated somatic mutations. By matching reads against an index of known variants, noise can be dramatically reduced, enabling the detection and quantification of those variants, even when they are present at less than 1% of the total sequenced population; this is comparable to, or better than, current diagnostic methods. Following the identification or exclusion of known variants, unmatched reads are grouped for BLAST searching to identify novel variants or contaminants. Known variants, novel variants, and contaminants were readily identified in tumor tissue using this approach. Our approach also enables an estimation of the per-base sequencing error rate, providing a confidence threshold for interpretation of the results in the clinic. This novel approach has immediate applicability to clinical testing for disease-associated genetic variants.
Leeds Institute of Molecular Medicine, Leeds University, Leeds, UK.
Philip A
PA
Chambers
Lucy F
LF
Stead
Joanne E
JE
Morgan
Ian M
IM
Carr
Kate M
KM
Sutton
Christopher M
CM
Watson
Victoria
V
Crowe
Helen
H
Dickinson
Paul
P
Roberts
Clive
C
Mulatero
Matthew
M
Seymour
Alexander F
AF
Markham
Paul M
PM
Waring
Philip
P
Quirke
Graham R
GR
Taylor
D045744:Cell Line, Tumor; D015179:Colorectal Neoplasms / Q000235:genetics / Q000473:pathology; D004252:DNA Mutational Analysis / Q000379:methods*; D020022:Genetic Predisposition to Disease / Q000235:genetics; D045325:HCT116 Cells; D018922:HL-60 Cells; D019073:HT29 Cells; D006801:Humans; D061986:MCF-7 Cells; D025202:Molecular Diagnostic Techniques / Q000379:methods*; D009154:Mutation; D016133:Polymerase Chain Reaction / Q000379:methods*; D011518:Proto-Oncogene Proteins / Q000235:genetics; D016283:Proto-Oncogene Proteins p21(ras); D015203:Reproducibility of Results; D017422:Sequence Analysis, DNA / Q000379:methods*; D018631:ras Proteins / Q000235:genetics
C117307:KRAS protein, human; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins