For PCR and sequencing analysis, nail and skin samples (approx.2 mm 2 mm 1 mm, ca. 10 mg) were frozen for 1 h at 150 8Cor 5 min in liquid nitrogen. Frozen samples were ground in a Multibeads Shocker (Yasui Kikai, Osaka, Japan) with metal cones for 3 min. Then the powdered samples were extracted with phenol– chloroform, as described elsewhere [13]. Ethachinmate coprecipitation reagent (3 mL, Nippon Gene, Tokyo, Japan) was applied to each sample at the beginning of isopropanol precipitation for maximum recovery of DNA