title: Shigenobu River Macroinvertebrate 454-pyrosequencing Reads
design: All specimens collected per study site were pooled, and dry weight (DW) was measured using a UMX2 Ultra-microbalance (Mettler-Toledo, Inc., USA). Based on the measured DW (19  403 mg per site), the samples were separated into 10 mg DW portions and placed in 1.5-ml tubes. The samples were then homogenized in the tubes using pestles. DNA was extracted using DNeasy Blood and Tissue Kit (Qiagen GmbH, Hilden, Germany) following the manufacturers instructions. Extracted DNA quantity and quality were measured using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington, DE, USA). DNA originating from the same study site were mixed with equal amounts of volume. A 658-bp fragment of the Cytochrome Oxidase I (COI) DNA barcode region was amplified using the universal primers LCO-1490 and HCO-2198 (Folmer et al., 1994), which both had 454-specific fusion primers (11-mer) and a 4-mer key sequence (TCAG) added to the 5-end. To pool multiple PCR products from the 10 study sites in one 454 pyrosequence run, a Molecular Identifier (MID) tag (6-mer) was also added to the 5-end of the LCO-1490 primer. PCR was conducted with a 40-l reaction volume containing 20 l of Phusion High-Fidelity PCR Master Mix with hydrofluoric acid (HF) buffer (New England Biolabs, UK), 2 l of each PCR primer (10 uM), 3 l of template DNA, and 13l of PCR-grade water using a T100TM Thermal Cycler (Bio-Rad Laboratories, CA, USA). The PCR conditions were denaturation at 94C for 2 min; 30 amplification cycles at 94C for 30 s, 40C for 30 s, and 72C for 60 s; and final extension at 72C for 10 min. Two (duplicate) PCR products were generated for each site. The resulting 20 PCR products were visualized using gel electrophoresis, and the target bands of successful samples were sliced and purified using the QIAquick Gel Extraction Kit (QIAGEN, Germany). The DNA concentration of each purified PCR product was measured with a Quantus Fluorometer (Promega, Wi., USA) using the QuantiFluor dsDNA System (Promega, Wi., USA) and was mixed with an equal molar amount. Pyrosequencing was carried out by Hokkaido System Science Co., Ltd. (Sapporo, Japan) using the 1/16 region of a GS FLX Titanium instrument (454; Roche Diagnostics).