Subsampled Assemblies and Hybrid Nucleotide Composition/Differential Coverage Binning for Genome-Resolved Metagenomics.

Metagenomic analyses for reconstruction of genomes from mixed microbial community datasets now routinely allow rapid, accurate genome recovery for tens to hundreds of organisms from environmental samples. This chapter provides a step-by-step protocol for reconstructing genomes from metagenomic datasets, with a focus on the most abundant community members. Subsampling assembly approaches are implemented to improve assembly of abundant genome sequences, an iterative process that targets progressively less abundant populations and improves total community representation in the final merged assembly. A hybrid approach to genome binning is described, combining differential coverage information from a series of metagenomic samples with nucleotide composition information. This approach strengthens binning through application of multiple independent variables for contig clustering. Genome curation through error correction and gap closure leads to high-quality draft genomes, and, for some community members, closed and complete genome sequences reconstructed directly from environmental samples.