TABLE 1 .

Key differences among open and closed high-throughput platforms for microbial community analysis^a

Step or parameter	Characteristic or consideration	Description of characteristic or consideration in indicated type of analysisb					Comments
		Open format			Closed format
		TGS	SMS	MTS	FGAs	PGAs
Sample preparation and analysis	Sample/target preparation	Complicated	Simple	Very complicated	Simple	Simple	DNA/RNA quality is important for all approaches
	Analysis of multiplex samples per assay	Large potential	Medium potential	Medium potential	Low (only one or two)	Low (only one or two)	FGAs and PGAs use 1 or 2 dyes for labeling, and it is difficult to multiplex samples in a single assay
	PCR amplification or whole-genome analysis	Yes	No	No	No/yes	Yes/no	Amplification introduces major problems for quantification
	Potential uneven hybridization	NA	NA	NA	Yes	Yes	Signal normalization is needed within and between arrays to correct signal differences due to systematic errors
Data processing and analysis	Raw data processing	Relatively easy	Difficult	Difficult	Easy	Easy	A major challenge for SMS and MTS with large raw datasets
	Phylogeny	Yes	Some	Some	No/yes	Yes	GeoChip uses gyrB for phylogeny
	Taxonomic resolution	Strain, species, genus	Strain, species	Strain, species	Strain, species	Genus, family	It depends on molecular markers with high resolution for functional genes
	Functional features	No/yes	Yes	Yes	Yes	No	TGS can analyze DNA and RNA for functional genes
	Signal threshold	Yes	NA	NA	Yes	Yes	Both PGAs and FGAs require a threshold to call positive signals, which is more or less arbitrary. Thus, some ambiguity exists for positive or negative spots.
	Requires a priori knowledge	No/yes	No	No	Yes	Yes	Closed-format technologies are designed based on known sequences
	Analysis of α diversity	Very good	Good	Very poor	Fair	Fair	Here, α diversity estimation is based on a single gene
	Data comparison across samples	Moderate	Difficult	Difficult	Easy	Easy	Random or undersampling is a major issue for open-format approaches
Performance	Coverage/breadth (no. of different genes detected)	Very low	High	High	High	Very low	TGS can analyze phylogenetic or functional genes
	Sampling depth (no. of sequences or OTUs per gene)	Very high	Low/medium	Low/medium	Medium	High	The sampling depth for closed-format approaches depends on the number of probes used
	Detection of rare species/genes	Medium	Difficult	Difficult	Easy	Easy	Easy for closed format as long as the appropriate probes are present
	Quantification	Low	Not known	Not known	High	Low/medium	Not rigorously tested for SMS and MTS; for PhyloChip, if RNA is used instead of DNA (no PCR step), quantification is high
	Susceptibility to the artifacts associated with random sampling process	Medium	High	High	Low	Medium/low	A major problem for sequencing approaches; PCR amplification may be involved in PhyloChip
	Potential discovery of novel genes/species	Yes	Yes	Yes	No	No
	Results skewed by dominant populations	Yes	Yes	Yes	No	No
	Sensitivity to (host) DNA/RNA contamination	No/yes	Yes	Yes	No	No	Difficult to remove host DNA/RNA contamination
Applicability and cost	Most promising applications	In-depth studies of microbial diversity or specific functional groups and discovery of novel genes	Surveys of microbial genetic diversity of unknown communities and discovery of novel genes	Surveys of functional activity of unknown microbial communities and discovery of novel genes	Comparisons of functional diversity and structure of microbial communities across many samples	Comparisons of taxonomic or phylogenetic diversity and structure of microbial communities across many samples	The choice of technology mainly depends on the biological questions and hypotheses to be addressed
	Relative cost per assay	Medium	High	High	Low	Low	It is challenging to make general statements of cost because they depend on technology platforms, depth of analysis, and approaches used for processing and analyzing data
	Cost per sample ($)	30–150	1200–4000	1500–4500	150–800	150–1000	This is only based on the cost of materials for target gene amplicon preparations and sequencing.
	Cost for bioinformatic analysis	Medium	High	High	Low	Low

^aSince various technologies have different features, it is difficult to make straightforward, point-by-point direct comparison. Thus, our attempt is to highlight the major differences of various technologies in a general sense. We attempt to focus on the issues important to microbial ecology within the context of environmental applications and complex microbial communities like those in soil rather than list the differences of various technologies in a comprehensive manner.

^bTGS, target gene (e.g., 16S rRNA, amoA, nifH) sequencing; SMS, shotgun metagenome sequencing; MTS, metatranscriptome sequencing; FGAs, functional gene arrays: the listed analysis is mostly based on GeoChip; PGAs, phylogenetic gene arrays: the listed analysis is mostly based on PhyloChip; NA, not applicable.