A quantitative SMRT cell sequencing method for ribosomal amplicons.
Advances in sequencing technologies continue to provide unprecedented opportunities to characterize microbial communities. For example, the Pacific Biosciences Single Molecule Real-Time (SMRT) platform has emerged as a unique approach harnessing DNA polymerase activity to sequence template molecules, enabling long reads at low costs. With the aim to simultaneously classify and enumerate in situ microbial populations, we developed a quantitative SMRT (qSMRT) approach that involves the addition of exogenous standards to quantify ribosomal amplicons derived from environmental samples. The V7-9 regions of 18S SSU rDNA were targeted and quantified from protistan community samples collected in the Ross Sea during the Austral summer of 2011. We used three standards of different length and optimized conditions to obtain accurate quantitative retrieval across the range of expected amplicon sizes, a necessary criterion for analyzing taxonomically diverse 18S rDNA molecules from natural environments. The ability to concurrently identify and quantify microorganisms in their natural environment makes qSMRT a powerful, rapid and cost-effective approach for defining ecosystem diversity and function.
Department of Earth and Environmental Sciences, Rutgers University-Newark, Newark, NJ 07102, United States. Electronic address: bethan.jones@science.oregonstate.edu.
Bethan M
BM
Jones
Department of Earth and Environmental Sciences, Rutgers University-Newark, Newark, NJ 07102, United States.
Adam B
AB
Kustka
D004247:DNA / Q000302:isolation & purification; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000145:classification / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics*; D017753:Ecosystem; D004783:Environmental Microbiology; D056890:Eukaryota / Q000145:classification / Q000235:genetics; D014644:Genetic Variation; D059014:High-Throughput Nucleotide Sequencing / Q000379:methods*; D064307:Microbiota / Q000235:genetics*; D009792:Oceans and Seas; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012337:RNA, Ribosomal, 18S / Q000235:genetics; D012623:Seawater / Q000382:microbiology; D017422:Sequence Analysis, DNA / Q000379:methods
D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S; D012337:RNA, Ribosomal, 18S; D004247:DNA
18S; Quantitative; Ribosomal; Ross Sea; SMRT; Sequencing; Silicoflagellate; qSMRT