Table 1. Overview of culture-independent genome sequencing methodologies applied to Chlamydiae to date.

CRB, Chlamydia-related bacteria; IFU, infection-forming units; IMS, immunomagnetic separation; LPS, lipopolysaccharide; MDA, multiple displacement amplification; WGA, whole genome amplification.

Culture-independent approach	Non-targeted (meta)genome capture				Targeted genome capture
Method	MDA	Depletion-enrichment	Cell-sorting-MDA	IMS-MDA		Sequence capture	Multiplexed microdroplet PCR
Molecular basis of method (also see Fig. 1 for schematic diagram)	Isothermal strand-displacing whole genome amplification of total genomic DNA	Depletion of host methylated DNA coupled with WGA of microbial DNA supernatant by MDA	Fluorescence-assisted cell sorting coupled with WGA of isolated single cells of interest (identified by PCR)	Anti-LPS antibody binding coupled with WGA by MDA		Biotinylated RNA probe hybridization to pathogen genome ‘bait’	Microdroplet PCR amplification of multiple DNA fragments spanning the target genome
Sample type(s) applied to (also see Fig. 2 for method selection)	Swab	Swab, tissue	Environmental samples	Swab stored in transport media		Swab, tissue, urine	Swabs, urine
Sample preparation stage at which to apply method	After total DNA extraction	After total DNA extraction	Initial sample, prior to DNA extraction	Initial sample, prior to DNA extraction		After total DNA extraction	After total DNA extraction
Discovery of novel species?	Yes	Yes	Yes	No		No	No
Detection of multiple strains/co-infection?	Yes	Yes	No	Yes		Yes	Yes
Cost per sample*	$	$	$$$$	$		$$¶	$$$
Speed* (hands on time)	<1 h§	~3–4 h§	~30 min – 2 h§	~5 h§		<1 h	~1–3 h
Sensitivity†	Ct value 25||	~1×103 genome copies per section	315 cells [58]	4 IFU per swab [66]; 1.1×105 genome copies (post-IMS) [65]		~2×103 genome copies per swab [72]; 3.3×105 (urine), 5.5×106 (swab) [69]	Not reported
Specificity‡	99.6 %||	99 % [55]	n/a	Up to 95 % [66]		88 % [74]	99 %
Variant detection?	Yes	Yes	Yes	Yes		Yes	Yes
Throughput	Moderate	Low	Low	High		High	Moderate
Limiting factors(s)/requirement(s)	Relative abundance of microbial and eukaryotic DNA in sample	Relative abundance of microbial and eukaryotic DNA in sample	Cells intact; minimal sample treatment Sample must be cryopreserved Samples in aquatic suspension are preferred	Cells intact; minimal sample treatment Suitable characterized surface-exposed antigen Sample must be preserved in transport media		Reference genome	Reference genome Specific bioinformatics software required
Chlamydial species applied to	C. trachomatis	Ca. C. sanzinia, Ca. C. corallus, Ca. Similichlamydia epinephelii	Novel CRB species	C. trachomatis		C. trachomatis, C. pecorum, C. pneumoniae, C. psittaci	C. trachomatis
Reference(s)	[50]	[55–57]	[58]	[64, 65]		[69, 72, 74, 75]	[77]

*Excluding DNA extraction and sequencing; relative cost to each other.

†Lowest amount of chlamydial DNA for 100 % genome coverage.

‡Highest percentage of non-chlamydial reads still allowing 90–100 % chlamydial genome coverage; will differ depending on sequencing platform and degree of multiplexing.

§MDA amplification step not included in time.

||Only 85 % of genome covered at least once.

¶Cost estimate for 1 : 1 bait/sample ratio.