Metagenome Sequencing of Anaerobic Digester Sludge

Three biomass samples were taken at weeks 1, 20, and 40 from a year long sampling campaign, directly from an AD reactor digesting food waste by the facility operators and shipped in ice-cooled containers to the University of Warwick. Upon receipt, they were stored at 4°C and then sampled into several 1–5mL aliquots within a few days and stored in 1.8 mL Cryovials at −80°C. Samples were defrosted at 4°C overnight prior to DNA extraction. DNA was extracted from a starting mass of 250 mg of anaerobic digester sludge using the MP Biomedical FastDNA SPIN Kit for Soil (cat no: 116560200) and a modified manufacturers protocol (see [27 (link)] for detailed protocol).
DNA size was assessed using a FemtoPulse (Agilent). The Pacific Biosciences protocol ‘Preparing 10 kb Library Using SMRTbell R Express Template Prep Kit 2.0 for Metagenomics Shotgun Sequencing’ was used to create libraries from 1.5 micrograms of DNA. In most cases the DNA was already 10 kb or smaller. Sample AD2W40 was a bit larger so the DNA was sheared using a g-TUBE (Covaris) for one library and unsheared for a second library. Libraries were not pooled due to the large number of reads desired. Sequencing was performed using a Sequel II sequencer (Pacific Biosciences) using version 8M SMRT cells and version 2.0 sequencing reagents with 30-hour movies with 2 hr pre-extension time to generate CCS reads.