June 1, 2021  |  

SMRT Sequencing and assembly of the human microbiome project Mock Community sample – a feasibility project.

While the utility of Single Molecule, Real-Time (SMRT) Sequencing for de novo assembly and finishing of bacterial isolates is well established, this technology has not yet been widely applied to shotgun sequencing of microbial communities. In order to demonstrate the feasibility of this approach, we sequenced genomic DNA from the Microbial Mock Community B of the Human Microbiome Project

June 1, 2021  |  

An interactive workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data.

The data throughput of next-generation sequencing allows whole microbial communities to be analyzed using a shotgun sequencing approach. Because a key task in taking advantage of these data is the ability to cluster reads that belong to the same member in a community, single-molecule long reads of up to 30 kb from SMRT Sequencing provide a unique capability in identifying those relationships and pave the way towards finished assemblies of community members. Long reads become even more valuable as samples get more complex with lower intra-species variation, a larger number of closely related species, or high intra-species variation. Here we present a collection of tools tailored for PacBio data for the analysis of these fragmented metagenomic assembles, allowing improvements in the assembly results, and greater insight into the communities themselves. Supervised classification is applied to a large set of sequence characteristics, e.g., GC content, raw-read coverage, k-mer frequency, and gene prediction information, allowing the clustering of contigs from single or highly related species. A unique feature of SMRT Sequencing data is the availability of base modification / methylation information, which can be used to further analyze clustered contigs expected to be comprised of single or very closely related species. Here we show base modification information can be used to further study variation, based on differences in the methylated DNA motifs involved in the restriction modification system. Application of these techniques is demonstrated on a monkey intestinal microbiome sample and an in silico mix of real sequencing data from distinct bacterial samples.

June 1, 2021  |  

Developments in PacBio metagenome sequencing: Shotgun whole genomes and full-length 16S.

The assembly of metagenomes is dramatically improved by the long read lengths of SMRT Sequencing. This is demonstrated in an experimental design to sequence a mock community from the Human Microbiome Project, and assemble the data using the hierarchical genome assembly process (HGAP) at Pacific Biosciences. Results of this analysis are promising, and display much improved contiguity in the assembly of the mock community as compared to publicly available short-read data sets and assemblies. Additionally, the use of base modification information to make further associations between contigs provides additional data to improve assemblies, and to distinguish between members within a microbial community. The epigenetic approach is a novel validation method unique to SMRT Sequencing. In addition to whole-genome shotgun sequencing, SMRT Sequencing also offers improved classification resolution and reliability of metagenomic and microbiome samples by the full-length sequencing of 16S rRNA (~1500 bases long). Microbial communities can be detected at the species level in some cases, rather than being limited to the genus taxonomic classification as constrained by short-read technologies. The performance of SMRT Sequencing for these metagenomic samples achieved >99% predicted concordance to reference sequences in cecum, soil, water, and mock control investigations for bacterial 16S. Community samples are estimated to contain from 2.3 and up to 15 times as many species with abundance levels as low as 0.05% compared to the identification of phyla groups.

June 1, 2021  |  

A workflow for the analysis of contigs from the metagenomic shotgun assembly of SMRT Sequencing data

The throughput of SMRT Sequencing and long reads allows microbial communities to be analyzed using a shotgun sequencing approach. Key to leveraging this data is the ability to cluster sequences belonging to the same member of a community. Long reads of up to 40 kb provide a unique capability in identifying those relationships, and pave the way towards finished assemblies of community members. Long reads are highly valuable when samples are more complex and containing lower intra-species variation, such as a larger number of closely related species, or high intra-species variation. Here, we present a collection of tools tailored for the analysis of PacBio metagenomic assemblies. These tools allow for improvements in the assembly results, and greater insight into the complexity of the study communities. Supervised classification is applied to a large set of sequence characteristics (e.g. GC content, raw read coverage, k-mer frequency, and gene prediction information) and to cluster contigs from single or highly related species. Assembly in isolation of the raw data associated with these contigs is shown to improve assembly statistics. A unique feature of SMRT Sequencing is the availability to leverage simultaneously collected base modification / methylation data to aid the clustering of contigs expected to comprise a single or very closely related species. We demonstrate the added value of base modification information to distinguish and study variation within metagenomic samples based on differences in the methylated DNA motifs involved in the restriction modification system. Application of these techniques is demonstrated on a mock community and monkey intestinal microbiome sample.

June 1, 2021  |  

Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron Mine.

Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in particular methylotrophic methanogenesis. Our results highlight the promising potential for long reads in metagenomic surveys of low-diversity environments.

June 1, 2021  |  

Profiling complex population genomes with highly accurate single molecule reads: cow rumen microbiomes

Determining compositions and functional capabilities of complex populations is often challenging, especially for sequencing technologies with short reads that do not uniquely identify organisms or genes. Long-read sequencing improves the resolution of these mixed communities, but adoption for this application has been limited due to concerns about throughput, cost and accuracy. The recently introduced PacBio Sequel System generates hundreds of thousands of long and highly accurate single-molecule reads per SMRT Cell. We investigated how the Sequel System might increase understanding of metagenomic communities. In the past, focus was largely on taxonomic classification with 16S rRNA sequencing. Recent expansion to WGS sequencing enables functional profiling as well, with the ultimate goal of complete genome assemblies. Here we compare the complex microbiomes in 5 cow rumen samples, for which Illumina WGS sequence data was also available. To maximize the PacBio single-molecule sequence accuracy, libraries of 2 to 3 kb were generated, allowing many polymerase passes per molecule. The resulting reads were filtered at predicted single-molecule accuracy levels up to 99.99%. Community compositions of the 5 samples were compared with Illumina WGS assemblies from the same set of samples, indicating rare organisms were often missed with Illumina. Assembly from PacBio CCS reads yielded a contig >100 kb in length with 6-fold coverage. Mapping of Illumina reads to the 101 kb contig verified the PacBio assembly and contig sequence. These results illustrate ways in which long accurate reads benefit analysis of complex communities.

June 1, 2021  |  

High-resolution evaluation of gut microbiota associated with intestinal maturation in early preterm neonates

Leaky gut, or intestinal barrier immaturity with elevated intestinal permeability, is the proximate cause of susceptibility to necrotizing enterocolitis in preterm neonates. We recently revealed intestinal barrier maturation was associated with exclusive breastfeeding, less antibiotic exposure, most importantly, altered composition of the gut microbiota. However, sequencing short regions of 16S rRNA gene amplicon failed to identify the specific bacterial groups associated with improved or aberrant intestinal permeability. In this study, we performed high-throughput amplicon sequencing of the full length 16S rRNA gene with single-nucleotide resolution for a cohort of 66 preterm neonates born at 24-33 weeks of gestation who had stool collected daily for 21 postnatal days. We assessed their intestinal permeability by measuring urine non-metabolized sugar probes lactulose and rhamnose during the first 7-10 days of life. We observed that intestinal barrier maturation was positively correlated with changes in specific amplicon sequence variants of species of Clostridiales and Bifidobacterium, while leaky gut was associated with specific strains of Escherichia coli. These results are promising in that they support the use of stool microbial biomarkers for the rapid, non-invasive, and cost-effective assessment of intestinal maturation in neonates.

June 1, 2021  |  

Metagenomic analysis of type II diabetes gut microbiota using PacBio HiFi reads reveals taxonomic and functional differences

In the past decade, the human microbiome has been increasingly shown to play a major role in health. For example, imbalances in gut microbiota appear to be associated with Type II diabetes mellitus (T2DM) and cardiovascular disease. Coronary artery disease (CAD) is a major determinant of the long-term prognosis among T2DM patients, with a 2- to 4-fold increased mortality risk when present. However, the exact microbial strains or functions implicated in disease need further investigation. From a large study with 523 participants (185 healthy controls, 186 T2DM patients without CAD, and 106 T2DM patients with CAD), 3 samples from each patient group were selected for long read sequencing. Each sample was prepared and sequenced on one Sequel II System SMRT Cell, to assess whether long accurate PacBio HiFi reads could yield additional insights to those made using short reads. Each of the 9 samples was subject to metagenomic assembly and binning, taxonomic classification and functional profiling. Results from metagenomic assembly and binning show that it is possible to generate a significant number of complete MAGs (Metagenome Assembled Genomes) from each sample, with over half of the high-quality MAGs being represented by a single circular contig. We show that differences found in taxonomic and functional profiles of healthy versus diabetic patients in the small 9-sample study align with the results of the larger study, as well as with results reported in literature. For example, the abundances of beneficial short- chain fatty acid (SCFA) producers such as Phascolarctobacterium faecium and Faecalibacterium prausnitzii were decreased in T2DM gut microbiota in both studies, while the abundances of quinol and quinone biosynthesis pathways were increased as compared to healthy controls. In conclusion, metagenomic analysis of long accurate HiFi reads revealed important taxonomic and functional differences in T2DM versus healthy gut microbiota. Furthermore, metagenome assembly of long HiFi reads led to the recovery of many complete MAGs and a significant number of complete circular bacterial chromosome sequences.

April 21, 2020  |  

A microbial factory for defensive kahalalides in a tripartite marine symbiosis.

Chemical defense against predators is widespread in natural ecosystems. Occasionally, taxonomically distant organisms share the same defense chemical. Here, we describe an unusual tripartite marine symbiosis, in which an intracellular bacterial symbiont (“Candidatus Endobryopsis kahalalidefaciens”) uses a diverse array of biosynthetic enzymes to convert simple substrates into a library of complex molecules (the kahalalides) for chemical defense of the host, the alga Bryopsis sp., against predation. The kahalalides are subsequently hijacked by a third partner, the herbivorous mollusk Elysia rufescens, and employed similarly for defense. “Ca E. kahalalidefaciens” has lost many essential traits for free living and acts as a factory for kahalalide production. This interaction between a bacterium, an alga, and an animal highlights the importance of chemical defense in the evolution of complex symbioses.Copyright © 2019 The Authors, some rights reserved; exclusive licensee American Association for the Advancement of Science. No claim to original U.S. Government Works.

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