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Mutation Detection by Clonal Sequencing of PCR Amplicons and Grouped Read Typing is Applicable to Clinical Diagnostics


  • Communicated by Paolo M. Fortina

  • Contract grant sponsors: Leeds CR-UK Centre Award (to G.R.T.); Yorkshire Cancer Research Program (to P.Q.); Leeds University Scholarship (to K.M.S.). Grant number C37059/A11941 from Cancer Research UK to PAC

Correspondence to Graham R. Taylor, Leeds Institute of Molecular Medicine and Leeds Teaching Hospitals, 6.2 Clinical Sciences Building, St James's University Hospital, Leeds LS9 7TF, UK. E-mail:


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.