Table 4.

Challenges for the integration of metagenomics into public health

Challenge	Description	Relevance	Solution	Reference(s)
Multiple technologies	Next-generation sequencing can be performed on multiple platforms each with different characteristics, and each constantly under improvement	Difficulty comparing results from different platforms and with those from older techniques	Pipelines must be constantly updated to account for new techniques	[74,76,92]
		Universal approach not yet possible	Different platforms should be utilized depending on the question asked
		Continuously evolving technology requires skilled workforce rather than established pipelines
Computational resources	Our ability to generate DNA sequence data has rapidly surpassed our computational abilities to analyze the data	Significant requirements for storage of DNA sequence	Perform analysis using a staged approach	[69,93]
		Assembling and identifying short reads from next-generation sequencing is computationally intensive	Cloud computing
Suitable reference databases	Multiple reference databases are available, which may generate different results depending on the database used	Certain features of a metagenomic sample might be missed if the wrong database is used	HMP aims to sequence multiple references genomes associated with the human body	[94]
		Limited by the diversity represented in each database	HMP currently has a total of 6,500 reference sequences generated
Short read lengths	Read lengths depend on sequencing platform used	Makes de novo assembly more complicated	Read lengths are continually increasing	[92,95]
		More difficult to identify large-scale genomic variations and repetitive regions	Third-generation sequencing platforms promise much longer read lengths
Causation	Finding a pathogen in a disease sample does not imply causation	Important to determine causation before changing public health management	Follow-up studies are required - for example, using animal models, or serological or epidemiological methods.	[11,75,96]
		False association can lead to costly, useless or even potentially harmful therapies	Results must be independently validated
Contamination	Metagenomics can detect contaminants from cell cultures, reagents and laboratory equipment	Contaminants may be incorrectly associated with the disease of interest	Negative controls must be used	[97]
			Researchers must consider the plausibility of the findings
			Results must be independently validated
Privacy	Host nucleic acids are almost always sequenced in metagenomics studies	Host genetic sequences are confidential	Host DNA to be available only to researchers in HMP	[92,98]
		Human subjects might be traceable from their DNA sequences	Only microbiome data are released to the public