title: LMG11-01 Cruise Station 600.040 400m water sample (Circumpolar Deep Water) Thaumarchaeota 16S rRNA
design: Thaumarchaeal 16S rRNA and archaeal amoA amplicons were sequenced from the Palmer LTER study region using a Roche 454 GS-FLX instrument (454 Life Sciences, Branford, CT) with Titanium chemistry (maintained by the Georgia Genomics Facility, University of Georgia, Athens, GA; http://dna.uga.edu/).  Thaumarchaeal 16S rRNA primers were tested for specificity using TestPrime 1.0 (http://www.arb-silva.de/search/testprime/) after Klindworth et al. (2013).  The first of the five 517F primers used to sequence the Archaea v6-v4 region (5’-GCCTAAAGCATCCGTAGC -3’) by the VAMPS project (http://vamps.mbl.edu/) was found to amplify 93.3% of Marine Group I Thaumarchaeota and only 11% of Euryarchaeota (with no mismatches allowed), and so was selected for this study (hereinafter referred to as 517Fa).  Primers 517Fa and 1058R (Archaea 16S rRNA; VAMPS project – http://vamps.mbl.edu/resources/primers.php) and CamoA-19f and CamoA-616r (Archaea amoA; (Pester et al., 2012, Env. Microbiol.) were modified with Titanium (Lib-L) adaptors and sample-specific barcodes.    Samples were amplified in triplicate using 1 µL of DNA extract, Q5® High-Fidelity DNA Polymerase (New England Biolabs, Ipswich, MA), and 1 µL of each primer with the following PCR conditions: initial denaturation at 98°C for 3 minutes; 30 cycles of denaturation at 98°C for 10 seconds, annealing at 63°C (16S rRNA) or 59°C (amoA) for 30 seconds, and extension at 72°C for 30 seconds; followed by a final extension at 72°C for 2 minutes.  PCR reactions were pooled following amplification and purified using the AMPure® XP PCR purification kit (Agencourt Bioscience, Beverly, MA) with a modified 1:1 volume of PCR product to AMPure® bead solution.  Purified amplicons were quantified using the Quant-iT™ PicoGreen® dsDNA Kit (Invitrogen, Carlsbad, CA), pooled in equimolar concentrations, and submitted for sequencing at the Georgia Genomics Facility.