Profiling Microbial Diversity in Sediment

DNA from ¹⁴N incubation samples #3731 T_0d, #3731 T_326d, and #5133 T_170d was extracted from ~0.5 g of sediment using the UltraClean Soil DNA isolation kit (Mo Bio Laboratories, Carlsbad, CA) and microbial community composition was profiled using the 515f (5′-GTGYCAGCMGCCGCGGTAA-3′) and 806r (5′-GGACTACNVGGGTWTCTAAT-3′) primer pair (Caporaso et al., 2011 (link)). PCR barcoding reactions were conducted in house at Caltech and amplicons were outsourced to Laragen Inc. (Laragen Inc., Culver City, CA) for sequencing on the Illumina MiSeq platform. Resulting sequence data consisted of 250 bp paired end reads, which were assembled into a single contig and analyzed using Qiime 1.8 (Caporaso et al., 2010 (link)). Sequences were trimmed to a minimum quality value of 30, and taxonomy was assigned to each OTU representative based on the SILVA 115 rRNA database uclust (Edgar et al., 2011 (link)) using a sequence similarity threshold of 99%. The full protocol is contained in a GNU makefile (Supplemental Data Sheet 1); the makefile must be in the same directory as the raw sequencing reads, and qiime 1.8 and usearch must be installed. The complete protocol can be run by typing “./orphanlab_itag_protocol.mk all” into the command prompt.