Ami Bhatt: Closing highly repetitive bacterial genomes from metagenomic nanopore sequencing
Ami Bhatt, Assistant Professor of Medicine (Hematology) and Genetics at Stanford University kicked off proceedings with a highly entertaining presentation describing her team’s latest research utilising nanopore sequencing to assemble complete genomes from complex microbial communities. In addition to understanding the microbial pathogenicity, Ami is particularly interested in how microorganisms may impact the outcomes of patients with cancer or cardiac disease. Ami explained that the human body contains approximately 10 trillion microbes – as many as there are cells in the human body. The sheer number, variety and complexity of microbes, combined with the high level of host DNA makes accurate genomic analysis of human microbiomes extremely challenging. Ami explained how this situation limits our understanding of gene content and regulation. Traditional techniques such as 16S rRNA gene sequencing and shotgun sequencing provide pieces of the puzzle but large gaps still remain.
To support their aim of generating complete genome assemblies from human metagenomic samples, the team at Stanford University evaluated a number of approaches, including a novel DNA partitioning approach using the Chromium platform combined with a bespoke assembly platform called Athena. In practice, the genomes of some microbial species assembled well with high contig N50 values; however, others, such as Prevotella spp (a dominant constituent of the gut microbiome of the Hadza community in Tanzania) assembled poorly. At 4800x coverage, Ami stated that sequencing depth was not the issue and instead suggested that the problem lay in the high level of genomic repeats found in these species.
Ami described how Eli Moss, a graduate student in her lab, whom she credits for the majority of this work came to her with the idea to use long-read nanopore sequencing to provide more complete genome assemblies. However, citing the challenge of obtaining high-molecular weight DNA from stool samples, Ami rejected his proposal. Undeterred, Eli spent 6 months testing and optimising various sample preparation methodologies – which Ami humorously described as “not a deeply pleasant task”. His perseverance paid off though as his technique, which comprises enzymatic cell wall degradation, phenol:chloroform extraction, gravity column purification and bead-based size selection, delivered both yield and molecular weight suitable for the generation of long sequencing reads.
Following library preparation using the Rapid Sequencing Kit, the DNA was sequenced using a MinION. A number of assembly tools were evaluated, with optimal results provided by Flye and Canu. Both of these tools allowed the assembly of the highly repetitive Prevotella copri genome in a single contig of 3.8 Mb – a result that Ami described as ‘breathtaking’. Not only was this achieved from metagenomic sequencing of stool samples – a significant challenge in itself – but it also represents the first full-length P. copri genome. This genome significantly improves on previous short-read based assembles of the organism.
Using the CheckM tool, the genic completeness was considered low; however, after refining their computational workflow and implementing sequence polishing using pilon a high level of completeness was obtained. The full computational workflow is freely available online for use by other researchers. In summarising her presentation, Ami commented that their method: ‘represents an effective, straightforward solution for the complete and efficient de novo characterization of structurally complex bacterial genomes within the gut microbiome’. The team now plan to assemble further microbial genomes from the metagenomic data and also analyse the data to explore methylation profiles (which is provided as standard in the raw nanopore sequencing data) to predict plasmid-chromosome pairing.