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Molecular methods used for microbiome analysis1

Method2 Description Characteristics Primary advantages Primary disadvantages
qPCR Amplifies and quantifies a targeted DNA molecule Dye or probe used to bind double-stranded DNA, which causes intensity of fluorescent emissions to increase Low cost; high sensitivity allows for detection of sequences at low concentrations Limited in scope
FISH Sensitive detection of specific nucleic acid sequences in metaphase or interphase cells Manual procedure of biological samples; fluorescence intensities measured using FLEX (a quantitative fluorescence microscope system) Allows for localization and study of spatial organization of cells as they occur in their natural habitat Costly; not easily scalable for disease screenings
RFLP High-throughput fingerprinting technique used to explore changes in structure and composition of microbial communities. DNA sample digested by restriction enzymes to characterize microbiota of specific regions. Fragments then separated according to length by gel electrophoresis. Provides a broad view of microbial systems Primers not specific
DGGE PCR-amplified 16S rRNA fragments separated on polyacrylamide gel containing gradient of denaturant (e.g., urea, formamide) Gel-based method of fingerprinting Provides a broad view of microbial systems Only semiquantitative and insensitive
TGGE PCR-amplified 16S rRNA fragments separated on polyacrylamide gel containing gradient of temperatures Gel-based method of fingerprinting Generate qualitative differences in microbial ecology Only semiquantitative and insensitive
454 sequencing Pyrosequencing light emission 400 to 600 base reads 16S coverage is good Cost limits shotgun coverage
Illumina sequencing Fluorescent, stepwise sequencing 100 to 150 base reads Very high coverage owing to high instrument output and very low cost Increased bioinformatics costs and time
Sanger sequencing Fluorescent, dideoxy terminator 750 base reads or greater High read length and accuracy Compared with next-generation sequencing, is costly and has low throughput
1Summarized from Pinkel et al. (1986), Muyzer et al. (1993), Liu et al. (1997), Zoetendal et al. (1998), Rigottier-Gois et al. (2003), Richards et al. (2005), Schütte et al. (2008), Petrosino et al. (2009), and Weinstock (2012).
2qPCR = quantitative PCR; FISH = fluorescent in situ hybridization; DGGE = denaturing gradient gel electrophoresis; TGGE = temperature gradient gel electrophoresis.