Table 1. Techniques used to investigate the human gut microbiota; advantages, disadvantages and examples of use.

	Microflora associated characteristics	Culture-dependent techniques	Culture-independent techniques	High-throughput sequencing
Technique description	The use of characteristics associated with microbes e.g., SCFA production to identify if differences exist in the gut microbial populations between different subject groups	Use of selective media to culture specific microorganisms or species of microorganisms e.g., Man Rogosa Sharpe (MRS) media for lactobacilli growth	Identify bacteria through isolation and amplification of bacterial DNA e.g., 16S rRNA gene. Includes: PCR, DGGE, TGGE, qPCR, dot blot hybridization, FISH, flow cytometry	Sequencing based approaches used to rapidly identify bacteria using bacterial DNA as template e.g., 454, Illumina, SoLID, Ion torrent
History of use	To date has been predominantly used as an initial population screen or in epidemiological studies	Historically, the most frequently used approach to identify bacteria present in various environments	Increasingly popular in past 2 decades with increasing availability of computer based technologies and software programs	Became commercially available at the beginning of the 21st century and becoming increasingly popular ever since
Advantages	Simple Inexpensive Suitable as an initial screen to test a novel hypothesis Useful for large population screens e.g., in epidemiology studies	Quick Inexpensive Limited skill required Limited equipment needed Useful as the initial screen before more detailed investigations	Relatively inexpensive Relatively simple More detailed results achievable	Less biased results Very detailed information Bacterial profile in complex environments e.g., gut microbiota can be identified Huge phylogenetic information provided Relatively quick
Disadvantages	Provides limited information No bacterial species identification possible	Up to 90% of bacteria non-culturable Provides limited information Need prior knowledge of bacteria to screen for Requires further tests for species identification	Prone to PCR bias Requires more sophisticated equipment and training on their use May need several methods in combination to get appropriate level of details in results	Extremely expensive Data handling requirements are significant Requires training on sample preparation and machine use and experience of interpreting results
Examples of studies efficiently using this technique	References 10–12	References 13, 14	Reference 15	References 2, 16
Future use in infant gut microbiota research	Most likely to be used to test novel hypotheses and to be followed up with more detailed techniques	Likely to become infrequently used and to be mainly used in combination with and verified by newer technologies	Likely to remain popular in the coming decade, but decrease thereafter as increased availability and use of sequencing approaches occurs	Increased use since the year 2000 as cost is decreasing and likely to become the main approach used in the future