Comparison of shotgun metagenomic sequencing with activity-guided and PCR-based functional metagenomics.

Methods of enzyme discovery	Shotgun metagenomic sequencing	Activity-guided screening	PCR-based screening
Pros	• Complete functional profile of an environment	• Can lead to detection of new enzymes or folds catalyzing known reactions	• Sensitive for low-abundance sequences
	• Genomic context and taxonomy obtained through binning/assembly	• Well-developed methods to screen for industrially-relevant enzymes, e.g., lipases, cellulases	• Detect variation within a single gene family at the level of single nucleotide changes
	• Higher accuracy achievable with proximity-guided assembly and long-read sequencing methods	• Inexpensive	• Relatively inexpensive
	• Can be combined with other meta-omics analyses	• Activity-forward method guarantees enzymes are active and express well in E. coli
	• Generally less biased than activity- and PCR-based methods
Cons	• High sequencing depth required to detect genes in low abundance	• Limited to genes and small to medium-sized gene clusters that are expressed in the screening host	• Requires conserved DNA motifs in target sequences
	• Computationally-intensive assembly and binning	• Typically limited to types of reactions that can be screened rapidly	• Not effective for detecting novel enzyme seqences or folds
	• Challenging to infer function from sequence alone	• Can requires specific high-throughput screening equipment	• Little to no taxonomic information
		• No taxonomic information	• PCR-bias against GC-rich sequences
		• Can only screen for one type of reaction/function at a time	Short reads make gene cluster context difficult to recover