454 GS Junior sequencing of SAMD00048625
Bulk microarthropod community samples (assumed to be those typically extracted from 100 mL of core-sampled soil via Tullgren funnel and stored in vials with ethanol as preservative (at a recommended ???20??C); consists of 100 to 1,000 animals comprising primarily Collembola and mites) were transferred to 1.5-mL PCR tubes. The preservative ethanol was removed prior to DNA extraction. Total DNA was extracted from the community samples using DNeasy Blood and Tissue Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions except that microarthropods were milled using a micro homogenizer (BioMasher II; NIP, Tokyo, Japan) in lysis buffer. The extracted DNA samples were quantified using a Qubit 2.0 Fluorometer with a Qubit dsDNA HS assay kit (Thermo Fisher Scientific). The target gene region (mtCOI or mt16S) was amplified for each community sample using the primer pair for mtCOI (Col_mtCOI_382F: 5???-acaggatgaacagtttayccncc-3???; Col_mtCOI_695R: 5???-gggtctcctcctccngcngg-3???) or mt16S (Col_mt16S_39F: 5???-accgtgcaaaggtagc-3???; Col_mt16S_523R: 5???-aagatagaaaccaacctggc-3???) tailed with four-base multiplex identifier (MID) tags at the 5???-end of each primer. PCR was performed in a 20-??l reaction volume using a KAPA HiFi HotStart ReadyMix kit (2-ng DNA template, 10 ??l of 2?? KAPA HiFi HotStart ReadyMix, 0.3 ??M of each primer; KAPA Biosystems, Wilmington, Massachusetts, USA). The PCR conditions were as follows: 95??C for 2 min; 15-25 cycles at 98??C for 20 s, 53??C for 30 s, and 72??C for 30 s, followed by a final extension step at 72??C for 1 min. To keep PCR amplification from reaching a plateau, the PCR cycles to be used were determined for each sample beforehand by a preliminary examination in which the band intensities of the PCR products from several distinct cycles (e.g., 15, 18, 21, and 24) were compared by gel electrophoresis to identify the range of cycles where PCR was yet to reach a plateau for the sample (we recommend fewer than three cycles of difference in the PCR cycles between the samples to be analyzed simultaneously). The PCR products were purified by size selection for the expected amplicon size using an SPRIselect reagent kit (200-500 bp for mtCOI; 300-700 bp for mt16S; Beckman Coulter, Brea, California, USA). The purified amplicons were checked with an Agilent 2100 Bioanalyzer with High Sensitivity DNA kit (Agilent Technologies, Santa Clara, California, USA) for evaluation of their size distributions and thereby quantification of their molecular concentration by Agilent 2100 Expert Software. The amplicons to be sequenced simultaneously in a sequencing run were pooled into molar concentrations equal between samples. The pooled amplicons were ligated with adapters for 454 technology using a GS Rapid Library Preparation Kit (Roche) to prepare a single library. After further purification by size selection using the SPRIselect reagent kit, the library was quantified with KAPA Library Quantification Kit (Kapa Biosystems). Sequencing of the library was performed with a 454 GS Junior sequencer (Roche).
RYUKYU
DRX051512