Mosaic Ends Tagmentation (METa) Assembly for Highly Efficient Construction of Functional Metagenomic Libraries.

ABSTRACT: Functional metagenomic libraries, physical bacterial libraries which allow the high-throughput capture and expression of microbiome genes, have been instrumental in the sequence-naive and cultivation-independent exploration of metagenomes. However, preparation of these libraries is often limited by their high DNA input requirement and their low cloning efficiency. Here, we describe a new method, mosaic ends tagmentation (METa) assembly, for highly efficient functional metagenomic library preparation. We applied tagmentation to metagenomic DNA from soil and gut microbiomes to prepare DNA inserts for high-throughput cloning into functional metagenomic libraries. The presence of mosaic end sequences in the resulting DNA fragments synergized with homology-based assembly cloning to result in a 300-fold increase in cloning efficiency compared to traditional blunt-cloning-based protocols. We show that compared to published libraries prepared by state-of-the-art protocols, METa assembly is on average ca. 20- to 200-fold more efficient and can prepare gigabase-sized libraries with as little as 200 ng of input DNA. We show the usefulness of METa assembly first by using a normative 5-μg mass of soil metagenomic DNA to prepare a 700-Gb library that allowed us to discover novel nourseothricin resistance genes and a potentially new mode of resistance to this antibiotic and second by using only 300 ng of goose fecal metagenomic DNA to prepare a 27-Gb library that captured numerous tetracycline and colistin resistance genes. METa assembly provides a streamlined, flexible, and efficient method for preparing functional metagenomic libraries, enabling new avenues of genetic and biochemical research into low-biomass or scarce microbiomes. IMPORTANCE Medically and industrially important genes can be recovered from microbial communities by high-throughput sequencing, but precise annotation is often limited to characterized genes and their relatives. Cloning a metagenome en masse into an expression host to produce a functional metagenomic library, directly connecting genes to functions, is a sequence-naive and cultivation-independent method to discover novel genes. The process of preparing these libraries is DNA greedy and inefficient, however. Here, we describe a library preparation method that is an order of magnitude more efficient and less DNA greedy. This method is consistently efficient across libraries prepared from cultures, a soil microbiome, and a goose fecal microbiome and allowed us to discover new antibiotic resistance genes and mechanisms. This library preparation method will potentially allow the functional metagenomic exploration of microbiomes that were previously off limits due to their rarity or low microbial biomass, such as biomedical swabs or exotic samples.

SUBMITTER: Crofts TS 

PROVIDER: S-EPMC8269240 | biostudies-literature |

REPOSITORIES: biostudies-literature