Chapter Thirteen - Preparation of High-Molecular Weight DNA and Metagenomic Libraries from Soils and Hot Springs

Abstract

Metagenomics has become an important tool for the characterization of microorganisms, as it is independent of their enrichment or cultivation in the laboratory. Its application has led to the discovery of metabolisms from widespread, yet uncharacterized organisms such as the ammonia-oxidizing archaea. Different approaches ranging from the generation of short sequence reads by direct use of high-throughput sequencing technologies to the construction and sequencing of large-insert DNA libraries are being employed. For these purposes, DNA of high quality needs to be prepared from an environmental sample, which is a particular challenge for soils and sediments. Here we describe the methods used for the isolation of high-molecular weight (hmw) DNA from soil and hot spring samples, the subsequent production of large-insert metagenomic libraries, and the analysis of the resulting genomic fragments. Detailed step-by-step procedures include (1) how to isolate good-quality hmw DNA from soils and mud; (2) how to prepare the DNA for cloning; (3) how to efficiently establish, grow, pick, replicate, and store the large-insert metagenomic fosmid library; and finally, (4) how to screen the library for genes of interest.