Mosaic ends tagmentation (METa) assembly for highly efficient construction of functional metagenomic libraries

Terence S. Crofts*, Alexander G. McFarland, Erica M. Hartmann

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review


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 highthroughput 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.

Original languageEnglish (US)
Article numbere00524-21
Issue number3
StatePublished - Jun 2021


  • Antibiotic resistance
  • Beta-lactamases
  • Colistin
  • Functional metagenomics
  • Microbiome
  • Nourseothricin
  • RRNA methyltransferase
  • Shotgun cloning
  • Streptothricin acetyltransferase
  • Tetracyclines

ASJC Scopus subject areas

  • Microbiology
  • Ecology, Evolution, Behavior and Systematics
  • Biochemistry
  • Physiology
  • Modeling and Simulation
  • Molecular Biology
  • Genetics
  • Computer Science Applications


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