Functional Metagenomics to Study Antibiotic Resistance
The construction and screening of metagenomic expression libraries has great potential to identify novel genes and their functions. Here, we describe metagenomic library preparation from fecal DNA, screening of libraries for antibiotic resistance genes (ARGs), massively parallel DNA sequencing of the enriched DNA fragments, and a computational pipeline for high-throughput assembly and annotation of functionally selected DNA.
Key wordsFunctional metagenomics Antibiotic resistance genes Resistome Functional selections Massively parallel DNA sequencing High-throughput assembly Profile HMM-based annotation PARFuMS Resfams
The original implementation of functional metagenomics selections for interrogating resistance genes was described by J. Handelsman and colleagues in 1998. We thank A. Moore and B. Wang for protocol optimization of high-throughput versions of this method presented in this manuscript, A. Reyes and K. Forsberg for computational implementation of PARFuMS, M. Gibson for development of the Resfams database, and members of the Dantas lab for helpful general discussions and for comments on the manuscript.
This work was supported in part by the NIH Director’s New Innovator Award (http://commonfund.nih.gov/newinnovator/), the National Institute of Diabetes and Digestive and Kidney Diseases (NIDDK:http://www.niddk.nih.gov/), and the National Institute of General Medical Sciences (NIGMS: http://www.nigms.nih.gov/), of the National Institutes of Health (NIH) under award numbers DP2DK098089 and R01GM099538 to G.D. The content is solely the responsibility of the authors and does not necessarily represent the official views of the funding agencies.
- 1.Levy SB, Marshall B (2004) Antibacterial resistance worldwide: causes, challenges and responses. Nat Med 10. doi: 10.1038/nm1145
- 3.Perron GG, Whyte L, Turnbaugh PJ, Goordial J, Hanage WP, Dantas G, Desai MM (2015) Functional characterization of bacteria isolated from ancient arctic soil exposes diverse resistance mechanisms to modern antibiotics. PloS One 10(3). doi: 10.1371/journal.pone.0069533
- 13.Riesenfeld CS, Department of Plant Pathology, Microbiology Doctoral Training Program a, Goodman RM, Department of Plant Pathology, Microbiology Doctoral Training Program a, Gaylord Nelson Institute for Environmental Studies UoWM, Madison, WI 53706, USA, Handelsman J, Department of Plant Pathology, Microbiology Doctoral Training Program a (2015) Uncultured soil bacteria are a reservoir of new antibiotic resistance genes. Environ Microbiol 6(9):981–989. doi: 10.1111/j.1462-2920.2004.00664.x
- 18.Zerbino DR (2010) Using the Velvet de novo assembler for short-read sequencing technologies. Current protocols in bioinformatics/editoral board, Andreas D Baxevanis [et al] CHAPTER:Unit-11.15. doi: 10.1002/0471250953.bi1105s31