title: Metagenomic sequencing of geoduck hatchery tank water
design: DNA was isolated from 0.22 m filters from a single conical from each pH treatment from days 5, 8, and 12 for pH 8.2 and days 1, 8, and 12 for pH 7.1. DNA was isolated with the DNeasy Blood and Tissue Kit (Qiagen), following a modified version of the Gram-Positive Bacteria protocol. Briefly, filters were unfolded and placed in 1.7 mL snap cap tubes and incubated with 400 l of Buffer Al and 50 L of proteinase K overnight at 56C. The next morning, 400 l of 100% ethanol was added to each sample and samples were eluted in 50 l Buffer AE. Samples were quantified on a Qubit 3.0 with the Qubit 1x dsDNA HS Assay Kit using 5 l of sample. Libraries were prepared for multiplexing using a Nextera DNA Flex Kit (Illumina) following the manufacturers protocol for DNA quantities <10 ng, with the following adjustments. Polymerase chain reaction (PCR) steps were performed in 200 l thin-walled PCR tubes with a PTC-200 thermalcylcer (MJ Research); magnetic separations were performed in 1.7 mL snap-cap tubes using a DynaMag2 (Invitrogen); the thermalcycler used was a PTC-200 (MJ Research). Libraries were quantified on a Qubit 3.0 with the Qubit 1x dsDNA HS Assay Kit (Invitrogen). Library quality was assessed on a 2100 Bioanalyzer with a High Sensitivity DNA Kit (Agilent). The average fragment size was ~530 bp. Metagenome libraries were pooled and sequenced on an Illumina HiSeqX, 151bp paired-end.