June 1, 2021  |  

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.


June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing.

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 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% accuracy can be efficiently generated for low amounts of input DNA. 10 ng of input DNA sequenced in 4 SMRT Cells would generate >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community, since SMRT Sequencing has been shown to have no sequence-context bias. Long read lengths mean that that it would be reasonable to expect a high number of the reads to include gene fragments useful for analysis.


June 1, 2021  |  

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of these organisms as models, however, is hampered by the lack of a well-assembled and annotated genome sequences, fundamental to modern genetic and molecular studies. Here we report the genomic sequence of Macrostomum lignano and an accompanying characterization of its transcriptome. The genome structure of M. lignano is remarkably complex, with ~75% of its sequence being comprised of simple repeats and transposon sequences. This has made high quality assembly from Illumina reads alone impossible (N50=222 bp). We therefore generated 130X coverage by long sequencing reads from the PacBio platform to create a substantially improved assembly with an N50 of 64 Kbp. We complemented the reference genome with an assembled and annotated transcriptome, and used both of these datasets in combination to probe gene expression patterns during regeneration, examining pathways important to stem cell function. As a whole, our data will provide a crucial resource for the community for the study not only of invertebrate evolution and phylogeny but also of regeneration and somatic pluripotency.


June 1, 2021  |  

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

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 >100,000 such reads. While throughput is low compared to second-generation sequencing, the reads are a true random sampling of the underlying community. Long read lengths translate to a high number of the reads harboring full genes or even full operons for downstream analysis. Here we present the results of circular-consensus sequencing on a mock metagenomic community with an abundance range of multiple orders of magnitude, and compare the results with both 16S and shotgun assembly methods. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows to elucidate meaningful information from the very low-abundance community members. For example, given the above low-input sequencing approach, a community member at 1/1,000 relative abundance would generate 100 1-2 kb sequence fragments having 99% consensus accuracy, with a high probability of containing a gene fragment useful for taxonomic classification or functional insight.


June 1, 2021  |  

Profiling the microbiome in fecal microbiota transplantation using circular consensus and Single Molecule, Real-Time Sequencing

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 in the resulting assembly. Circular-consensus, single molecule, real-time (SMRT®) Sequencing reads in the 1-3kb range, with >99% accuracy can be efficiently generated for low amounts of input DNA. 10 ng of input DNA sequenced in 4 SMRT Cells on the PacBio RS II would generate >100,000 such reads. While throughput is lower compared to short-read sequencing methods, the reads are a true random sampling of the underlying community since SMRT Sequencing has been shown to have very low sequence-context bias. With reads >1 kb at >99% accuracy it is reasonable to expect a high percentage of reads include gene fragments useful for analysis without the need for de novo assembly. Here we present the results of circular consensus sequencing for an individual’s microbiome, before and after undergoing fecal microbiota transplantation (FMT) in order to treat a chronic Clostridium difficile infection. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows us to profile low abundance community members at the species level. We also show that using shotgun sampling with long reads allows a level of functional insight not possible with classic targeted 16S, or short read sequencing, due to entire genes being covered in single reads.


June 1, 2021  |  

Low-input long-read sequencing for complete microbial genomes and metagenomic community analysis

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 3 days by standard laboratory personnel. This approach is particularly important for analysis of metagenomic communities, in which genomic DNA is often limited. From these samples, full-length 16S amplicons can be generated, prepped with the standard SMRTbell library prep protocol, and sequenced. Alternatively, a 2 kb sheared library, made from a few ng of input DNA, can also be used to elucidate the microbial composition of a community, and may provide information about biochemical pathways present in the sample. In both these cases, 1-2 kb reads with >99.9% accuracy can be obtained from Circular Consensus Sequencing.


June 1, 2021  |  

Workflow for processing high-throughput, Single Molecule, Real-Time Sequencing data for analyzing the microbiome of patients undergoing fecal microbiota transplantation

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 500 bp 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 in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-3 kb range, with >99% accuracy can be generated using the previous generation PacBio RS II or, in much higher throughput, using the new Sequel System. While throughput is lower compared to short-read sequencing methods, the reads are a true random sampling of the underlying community since SMRT Sequencing has been shown to have very low sequence-context bias. With single-molecule reads >1 kb at >99% consensus accuracy, it is reasonable to expect a high percentage of reads to include genes or gene fragments useful for analysis without the need for de novo assembly. Here we present the results of circular consensus sequencing for an individual’s microbiome, before and after undergoing fecal microbiota transplantation (FMT) in order to treat a chronic Clostridium difficile infection. We show that even with relatively low sequencing depth, the long-read, assembly-free, random sampling allows us to profile low abundance community members at the species level. We also show that using shotgun sampling with long reads allows a level of functional insight not possible with classic targeted 16S, or short read sequencing, due to entire genes being covered in single reads.


June 1, 2021  |  

Using the PacBio Sequel System to taxonomically and functionally classify metagenomic samples in a trial of patients undergoing fecal microbiota transplantation

Whole-sample shotgun sequencing can provide a more detailed view of a metagenomic community than 16S sequencing, but its use in multi-sample experiments is limited by throughput, cost and analysis complexity. While short-read sequencing technologies offer higher throughput, read lengthss less fewer than 500 bp will rarely cover a gene of interest, and necessitate assembly before further analysis. Assembling large fragments requires sampling each community member at a high depth, significantly increasing the amount of sequencing needed, and limiting the analysis of rare community members. Assembly methods also risk It is also possible to incorrectly combine combining sequences from different community members.


June 1, 2021  |  

SMRT-Cappable-seq reveals the complex operome of bacteria

SMRT-Cappable-seq combines the isolation of full-length prokaryotic primary transcripts with long read sequencing technology. It is the first experimental methodology to sequence entire prokaryotic transcripts. It identifies the transcription start site and termination site, thereby directly defines the operon structures genome-wide in prokaryotes. Applied to E.coli, SMRT-Cappable-seq identifies a total of ~2300 operons, among which ~900 are novel. Importantly, our result reveals a pervasive read-through of previous experimentally validated transcription termination sites. Termination read-through represents a powerful strategy to control gene expression. Taken together this data provides a first glance at the complexity of the ‘operome’ in bacteria and presents an invaluable resource for understanding gene regulation and function in bacteria.


June 1, 2021  |  

Full-length cDNA sequencing of prokaryotic transcriptome and metatranscriptome samples

Next-generation sequencing has become a useful tool for studying transcriptomes. However, these methods typically rely on sequencing short fragments of cDNA, then attempting to assemble the pieces into full-length transcripts. Here, we describe a method that uses PacBio long reads to sequence full-length cDNAs from individual transcriptomes and metatranscriptome samples. We have adapted the PacBio Iso-Seq protocol for use with prokaryotic samples by incorporating RNA polyadenylation and rRNA-depletion steps. In conjunction with SMRT Sequencing, which has average readlengths of 10-15 kb, we are able to sequence entire transcripts, including polycistronic RNAs, in a single read. Here, we show full-length bacterial transcriptomes with the ability to visualize transcription of operons. In the area of metatranscriptomics, long reads reveal unambiguous gene sequences without the need for post-sequencing transcript assembly. We also show full-length bacterial transcripts sequenced after being treated with NEB’s Cappable-Seq, which is an alternative method for depleting rRNA and enriching for full-length transcripts with intact 5’ ends. Combining Cappable-Seq with PacBio long reads allows for the detection of transcription start sites, with the additional benefit of sequencing entire transcripts.


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  |  

Unbiased characterization of metagenome composition and function using HiFi sequencing on the PacBio Sequel II System

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 addresses these challenges: HiFi reads. HiFi reads combine QV20 accuracy with long read lengths, eliminating the need for assembly for most metagenome applications, including gene discovery and metabolic pathway reconstruction. In fact, the read lengths and accuracy of HiFi data match or outperform the quality metrics of most metagenome assemblies, enabling cost-effective recovery of intact genes and operons while omitting the resource intensive and data-inefficient assembly step. Here we present the application of HiFi sequencing to both mock and human fecal samples using full-length 16S and shotgun methods. This proof-of-concept work demonstrates the unique strengths of the HiFi method. First, the high correspondence between the expected community composition,16S and shotgun profiling data reflects low context bias. In addition, every HiFi read yields ~5-8 predicted genes, without assembly, using standard tools. If assembly is desired, excellent results can be achieved with Canu and contig binning tools. In summary, HiFi sequencing is a new, cost-effective option for high-resolution functional profiling of metagenomes which complements existing short read workflows.


June 1, 2021  |  

Low-input single molecule HiFi sequencing for metagenomic samples

HiFi sequencing on the PacBio Sequel II System enables complete microbial community profiling of complex metagenomic samples using whole genome shotgun sequences. With HiFi sequencing, highly accurate long reads overcome the challenges posed by the presence of intergenic and extragenic repeat elements in microbial genomes, thus greatly improving phylogenetic profiling and sequence assembly. Recent improvements in library construction protocols enable HiFi sequencing starting from as low as 5 ng of input DNA. Here, we demonstrate comparative analyses of a control sample of known composition and a human fecal sample from varying amounts of input genomic DNA (1 ug, 200 ng, 5 ng), and present the corresponding library preparation workflows for standard, low input, and Ultra-Low methods. We demonstrate that the metagenome assembly, taxonomic assignment, and gene finding analyses are comparable across all methods for both samples, providing access to HiFi sequencing even for DNA-limited sample types.


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.


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