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The minimum number of clones necessary to sequence in order to obtain the maximum information about hepatitis C virus quasispecies: a comparison of subjects with and without liver cancer


Edward Tabor M.D., Division of Emerging and Transfusion Transmitted Diseases, Office of Blood Research and Review, Food and Drug Administration, 1401 Rockville Pike, HFM-300, Rockville, MD 20852-1448, USA. E-mail:


Summary.  Most studies of hepatitis C virus (HCV) quasispecies have reported the results of sequencing only three to five clones per sample. The possibility that sequencing so few clones might not provide a representative picture of the quasispecies present in a sample has never been evaluated. The present study was conducted to evaluate whether sequencing greater numbers of clones results in better information about the HCV quasispecies number and distribution, and to compare the HCV quasispecies in liver cancer cases and controls. RNA was extracted from serial serum samples from six subjects with HCV-associated liver cancer and 11 age- and sex-matched HCV-infected controls without liver cancer. The hypervariable region 1 (HVR1) of the HCV genome was amplified, cloned, and sequenced. For further studies of 12 serum samples from two liver cancer cases and two matched controls, successive groups of 10 additional clones were sequenced up to a total of 50 clones per serum sample. When only 10 clones were sequenced from each specimen, no consistent differences were seen between the number of HCV quasispecies in the six liver cancer cases and the 11 controls. However, sequencing 40 clones from each of 12 samples from two liver cancer cases and two controls revealed a greater number of quasispecies in liver cancer cases than in controls. Testing an additional 10 clones (50 clones per sample) did not significantly increase the number of quasispecies detected.