DNeasy PowerSoil HTP 96 Kit (384)

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A total of 17 bacterial isolates, including isolates of Brevibacterium sp., Corynebacterium stationis, Brachybacterium sp., Arthrobacter sp., Propionibacterium acnes, Bacillus sp., Staphylococcus equorum, Staphylococcus succinus, Streptococcus anginosus, Desulfovibrio sulfodismutans, Serratia sp., Halomonas sp., Psychrobacter sp., Pseudomonas fragi, Vibrio rumoiensis, Escherichia coli, and Aliivibrio fischeri, were collected and stored in a phosphate-buffered saline (PBS) solution. The optical density at 600 nm (OD600) was measured for all isolates, and the corresponding cell density was estimated. Sixteen of these microbes (all except A. fischeri) were diluted to a final density of 1e8 cells per ml in a single 50-ml conical vial and designated “source” organisms. The A. fischeri isolate was diluted to 1e6 cells per ml, designated the “sink” microbe, and stored in a single 50-ml conical vial. Both source and sink microbes were stored in a −80°C freezer until aliquots were made for extractions. In addition, a mock community was created using these isolates by combining equal volumes of all samples, which also served as a reference for accounting for processing biases. An additional isolate of Clostridium sp. was measured and aliquoted into 16 different 2-ml tubes to be used for barcode testing. For DNA extraction at UCSD, 100 μl of source and sink samples was aliquoted into 2 96-well DNA extraction robot plates and 96 2-ml bead-beating extraction tubes, as indicated in the diagram (Fig. 1a; see also Fig. S1 in the supplemental material). Following the Earth Microbiome Project protocol (2), the Qiagen PowerMag kit (catalog number 27500-4-EP) was used for robot extractions, while the Qiagen DNeasy PowerSoil kit (catalog number 12888-100) was used for “manual, single-tube” extractions. To test the effect of antifoam on reducing well-to-well contamination, we added 2 μl of an antifoam A concentrate (catalog number A5633-25G; Sigma-Aldrich) to half of each of the robot plates (Fig. 1b and ​andc).c). In addition to processing samples at UCSD, an additional 192 samples were plated (96 unique samples in duplicate) in a 96-well plate and 96 individual 2-ml bead-beating tubes and sent to Argonne National Laboratory in the same plate map scheme. The manual tube samples were processed using the Qiagen DNeasy PowerSoil kit (catalog number 12888-100), while the manual plate samples were processed using the Qiagen DNeasy PowerSoil HTP 96 kit (catalog number 12955-4).

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