16S rRNA amplicon sequencing of Guaymas Basin sediment
Amplification of bacterial and archaeal 16S rRNA genes was performed following the Earth Microbiome protocol (Thompson et al., 2017) using updated primers 515F (forward primer, 5-GTGYCAGCMGCCGCGGTAA-3) (Parada et al., 2016) and 806R (reverse primer, 5-GGACTACNVGGGTWTCTAAT-3) (Apprill et al., 2015). Polymerase chain reaction (PCR) was performed in a final volume of 50 L consisting of 20 L of template DNA, 20 L Invitrogen Platinum Taq II 2x Master Mix, 1 L of each primer at a concentration of 10 M, and 8 L nuclease-free water for freeze/thaw extracted cells, and 25 L consisting of 5 L of template DNA, 10 L Invitrogen Platinum Taq II 2x Master Mix, 0.5 L of each primer at a concentration of 10 M, and 9 L nuclease-free water for DNA extracted with the FastDNA Spin Kit for Soil. The thermocycler conditions were: 94 oC for 3 min, then 28 cycles of 94 oC for 45 sec, 50 oC for 60 sec, and 72 oC for 90 oC, followed by an elongation step of 72 oC for 10 min. A selection of PCR products was checked for the expected length on a 1% agarose gel. All products were then purified using AMPure XP beads following the manufacturers protocol with a final elution volume of 35 L. For Illumina sequencing, Illumina dual index barcodes and sequencing adapters were attached in a second PCR reaction. The final volume for this reaction was 25 L, consisting of 5 L of purified amplicon, 10 L Invitrogen Platinum Taq II 2x Master Mix, 0.5 L of each primer at a concentration of 10 M (i5 and i7 primers), and 9 L nuclease-free water. The thermocycler conditions were: 95 oC for 3 min, then 8 cycles of 95 oC for 30 sec, 55 oC for 30 sec, 72 oC for 30 sec, followed by an elongation step of 72 oC for 5 min. The PCR products were again purified using AMPure XP beads, and then were quantified in triplicate reactions using Quant-iT Picogreen dsDNA assay (Invitrogen) on a Biotek Synergy H1 Hybrid microplate reader. Purified amplicons were then pooled with 7.5 ng of DNA each, or up to 30 L if a sample had less than 7.5 ng of DNA. The pooled library was quantified with Qubit high sensitivity assay. Purification, quality assessment, and sequencing of pooled libraries was performed at the Molecular Research Core Facility at Idaho State University using Illuminas MiSeq chemistry for 2 x 250 bp paired end reads.