Highly accurate long reads – HiFi reads – with single-molecule resolution make Single Molecule, Real-Time (SMRT) Sequencing ideal for full-length 16S rRNA sequencing, shotgun metagenomic profiling, and metagenome assembly.
Background: Microbial ecology is reshaping our understanding of the natural world by revealing the large phylogenetic and functional diversity of microbial life. However the vast majority of these microorganisms remain poorly understood, as most cultivated representatives belong to just four phylogenetic groups and more than half of all identified phyla remain uncultivated. Characterization of this microbial ‘dark matter’ will thus greatly benefit from new metagenomic methods for in situ analysis. For example, sensitive high throughput methods for the characterization of community composition and structure from the sequencing of conserved marker genes. Methods: Here we utilize Single Molecule Real-Time (SMRT) sequencing…
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
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…
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…
Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. A 2 kb SMRTbell library only requires a few ng of gDNA when carrier DNA is added to the library. The resulting libraries can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes…
There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments generally use short-read, second-generation sequencing, which results in data processing difficulties. For example, reads less than 1 kb in length will likely not cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members…
High-throughput sequencing of the complete 16S rRNA gene has become a valuable tool for characterizing microbial communities. However, the short reads produced by second-generation sequencing cannot provide taxonomic classification below the genus level. In this study, we demonstrate the capability of PacBio’s Single Molecule, Real-Time (SMRT) Sequencing to generate community profiles using mock microbial community samples from BEI Resources. We also evaluate multiplexing capabilities using PacBio barcodes on pooled samples comprising heterogeneous 16S amplicon populations representing soil, fecal, and mock communities.
There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate…
There are many sequencing-based approaches to understanding complex metagenomic communities spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR. Whole-sample shotgun experiments generally use short-read sequencing, which results in data processing difficulties. For example, reads less than 500bp in length will rarely cover a complete gene or region of interest, and will require assembly. This not only introduces the possibility of incorrectly combining sequence from different community members, it requires a high depth of coverage. As such, rare community members may not be represented…
Microbial genome sequencing can be done quickly, easily, and efficiently with the PacBio sequencing instruments, resulting in complete de novo assemblies. Alternative protocols have been developed to reduce the amount of purified DNA required for SMRT Sequencing, to broaden applicability to lower-abundance samples. If 50-100 ng of microbial DNA is available, a 10-20 kb SMRTbell library can be made. The resulting library can be loaded onto multiple SMRT Cells, yielding more than enough data for complete assembly of microbial genomes using the SMRT Portal assembly program HGAP, plus base modification analysis. The entire process can be done in less than…
The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. One popular, efficient method of profiling communities is to amplify and sequence the evolutionarily conserved 16S rRNA sequence. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost and species-level classification is often not possible. SMRT Sequencing easily spans the entire 1.5 kb 16S gene, and in combination with highly-accurate single-molecule sequences, can improve the identification of individual species in a metapopulation. However, when amplifying…
The constituents and intra-communal interactions of microbial populations have garnered increasing interest in areas such as water remediation, agriculture and human health. Amplification and sequencing of the evolutionarily conserved 16S rRNA gene is an efficient method of profiling communities. Currently, most targeted amplification focuses on short, hypervariable regions of the 16S sequence. Distinguishing information not spanned by the targeted region is lost, and species-level classification is often not possible. PacBio SMRT Sequencing easily spans the entire 1.5 kb 16S gene in a single read, producing highly accurate single-molecule sequences that can improve the identification of individual species in a metapopulation.However,…
Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with short-read technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly requires high coverage for error correction, the benefits of unbiased coverage have in the past been lost for low abundance species. SMRT Sequencing performance improvements and the introduction of the Sequel II System has enabled a…
Recent work comparing metagenomic sequencing methods indicates that a comprehensive picture of the taxonomic and functional diversity of complex communities will be difficult to achieve with one sequencing technology alone. While the lower cost of short reads has enabled greater sequencing depth, the greater contiguity of long-read assemblies and lack of GC bias in SMRT Sequencing has enabled better gene finding. However, since long-read assembly typically requires high coverage for error correction, these benefits have in the past been lost for low-abundance species. The introduction of the Sequel II System has enabled a new, higher throughput, assembly-optional data type that…