Prokaryotic Community Profiling from Pore Fluids Collected from a Natural Gas Reservoir
One sample was processed in combination with Callister SERDP samples. Files have been trimmed to reflect only the singular sample. DNA was provided in a 1.5 mL tube and was below concentration detection using Qubit high sensitivity DNA kit. The sample was amplified in triplicate reactions of 50 L. The composition of PCR was as follows: First PCR: PCR reagents for amplifying target region v4 of 16S rRNA gene Platinum II 2X Master Mix: 25 L (for 1X final concentration) Forward Primer GTGYCAGCMGCCGCGGTAA (5 M): 2 L (for 0.2 M final concentration) Reverse Primer GGACTACNVGGGTWTCTAAT (5 M): 2 L (for 0.2 M final concentration) Nuclease free water: 16 L. DNA: 5 L. Thermocycler conditions using a ProFlex PCR System were as follows: PCR program for amplifying target region  27 cycles Hot start  94 C for 3 minutes Denature  94 C for 45 seconds Anneal  50 C for 60 seconds Extend  72 C for 90 seconds Final extension  72 C for 10 minutes Hold  4 C AMPure XP Bead Clean up: Beads were mixed with DNA at a ratio of 1.8X beads. Samples were washed twice with 150 L of 80 % ethanol. Final elution was performed in 40 L of nuclease free water, combining the beads for elution from the three replicates. This was done to concentrate any amplicon product generated. Second PCR: PCR reagents for applying barcodes for multiplexing Platinum II 2X Master Mix: 12.5 L (for 1X final concentration) Barcode i7 (2.5 M): 2.5 L (for 0.25 M final concentration) Barcode i5 (2.5 M): 2.5 L (for 0.25 M final concentration) Nuclease free water: 2.5 L Cleaned DNA: 5 L Barcode combination: i7_N703 and i5_S510 PCR program for applying barcodes for multiplexing  8 cycles Hot start  95 C for 3 minutes Denature  95 C for 30 seconds Anneal  55 C for 30 seconds Extend  72 C for 30 seconds Final extension  72 C for 5 minutes Hold  4 C AMPure XP Bead Clean up: Beads were mixed with DNA at a ratio of 1.8X beads. Samples were washed twice with 150 L of 80 % ethanol. Final elution was performed in 50 L of nuclease free water. Picogreen dsDNA quantification assay: Samples were quantified in triplicate using 1 L of DNA. Sampling pooling: According to the Picogreen quantification, 40 ng of DNA per sample was pooled into 15 mL tubes for each plate. Using the Zymo DNA clean and concentrator kit, sample pools were concentrated and combined into a single 1.5 mL tube containing 50 L of cleaned DNA. If a sample did not have high enough concentration to pool 40 ng, 40 L was transferred. The final concentration of the pooled sample was 2.75 ng/L (all samples). Illumina MiSeq sequencing: Flow cell name: 241017_M03018_0240_000000000-DNKMD Sample was diluted to 2nM and the standard Illumina library prep was followed to load the samples onto the MiSeq for v2 sequencing using a Nano 500 cycle flow cell with a 20 % PhiX spike-in. Raw fastq files are located on the MiSeq for a short amount of time or in the network/pnl/projects/miseq-data/_MiSeqDataPush directory under the corresponding flow cell name.