| Abstract | This protocol describes the laboratory steps required to capture cpn60 gene fragments from microbial community DNA obtained from an environment of interest. This approach facilitates the determination of the taxonomic composition of a sample, providing information that spans all Domains of life (Bacteria, Eukarya, and Archaea). This method is independent of amplification biases associated with PCR, and provides a microbial community profile that is consistent with that generated using whole community shotgun sequencing, with substantially less sequencing effort required for the same community coverage. The protocol involves shearing the whole community genomic DNA by sonication, adding Illumina index primers, then hybridizing the sheared DNA to biotinylated RNA probes that correspond to the entire cpn60 reference database. Hybrids are selected using magnetic streptavidin beads, washed, and sequenced using an Illumina MiSeq platform. The protocol involves approximately five hours of hands-on time per sample over two days, along with a 24 hour hybridization step. In this protocol we describe the major changes we have implemented using the manufacturer’s recommendations as a starting point. We also provide tips and expected results. |
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