Person: Alma E. Parada

Abstract

Abstract

Although shotgun metagenomic sequencing of microbiome samples enables partial reconstruction of strain-level community structure, obtaining high-quality microbial genome drafts without isolation and culture remains difficult. Here, we present an application of read clouds, short-read sequences tagged with long-range information, to microbiome samples. We present Athena, a de novo assembler that uses read clouds to improve metagenomic assemblies. We applied this approach to sequence stool samples from two healthy individuals and compared it with existing short-read and synthetic long-read metagenomic sequencing techniques. Read-cloud metagenomic sequencing and Athena assembly produced the most comprehensive individual genome drafts with high contiguity (>200-kb N50, fewer than ten contigs), even for bacteria with relatively low (20×) raw short-read-sequence coverage. We also sequenced a complex marine-sediment sample and generated 24 intermediate-quality genome drafts (>70% complete, <10% contaminated), nine of which were complete (>90% complete, <5% contaminated). Our approach allows for culture-free generation of high-quality microbial genome drafts by using a single shotgun experiment.

Abstract

Marine sediments harbor an estimated 50% of all microbes on earth. However, the identity, distribution, and metabolism of deep-sea benthic microbes are poorly understood. We collected sediment cores at 10 sites across 300km off the San Francisco Coast spanning 100-4500m water depth and 0-200cm sediment depth. Using amplicon sequencing of 16S rDNA and metagenomic sequencing, we investigated trends in microbial community composition across physicochemical gradients, and evaluated metabolic capabilities of detected microbes. Microbial community similarities associated with water and sediment depth, correlating to changes in pressure, temperature and oxygen concentrations of overlying water, as well as pore water ammonium, phosphate, and nitrate concentrations. Many microbes were from potentially novel lineages: an average of 34% of amplicon sequence variants (ASVs) present in each sample were <90% similar to the 16S rRNA gene of representative genomes, and up to 13% of ASVs found in one sample were <70% similar to representatives. Metagenomic sequencing of surface sediment from 3500m water depth using a novel assembly method combining 10X genomics and barcoded short reads resulted in 24 MAGs with >70% completion, <10% contamination, and a 16S rRNA gene. The 9 most complete MAGs had average amino acid identities <65% to their best 16S rDNA representative genome match. These MAGs indicate potential for sulfate and sulfite reduction, nitrification, and general heterotrophic growth. These results indicate microorganisms distantly related to those previously characterized, dominated our sediment samples and mediate important conversions in carbon, sulfur, and nitrogen cycles in this environment.