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Asset Tag: VM_MID_AM

June 1, 2021  |  

Multiplexing strategies for microbial whole genome SMRT Sequencing

The increased throughput of the RS II and Sequel Systems enables multiple microbes to be sequenced on a single SMRT Cell. This multiplexing can be readily achieved by simply incorporating a unique barcode for each microbe into the SMRTbell adapters after shearing genomic DNA using a streamlined library construction process. Incorporating a barcode without the requirement for PCR amplification prevents the loss of epigenetic information (e.g., methylation signatures), and the generation of chimeric sequences, while the modified protocol eliminates the need to build several individual SMRTbell libraries. We multiplexed up to 8 unique strains of H. pylori. Each strain was sheared, and processed through adapter ligation in a single, addition only reaction. The barcoded strains were then pooled in equimolar quantities, and processed through the remainder of the library preparation and purification steps. We demonstrate successful de novo microbial assembly and epigenetic analysis from all multiplexes (2 through 8-plex) using standard tools within SMRT Link Analysis using data generated from a single SMRTbell library, run on a single SMRT Cell. This process facilitates the sequencing of multiple microbial genomes in a single day, greatly increasing throughput and reducing costs per genome assembly.

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June 1, 2021  |  

Minimization of chimera formation and substitution errors in full-length 16S PCR amplification

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, this process still relies upon PCR amplification from a mixture of similar sequences, which may result in chimeras, or recombinant molecules, at rates upwards of 20%. These PCR artifacts make it difficult to identify novel species, and reduce the amount of informative sequences. We investigated multiple factors that may contribute to chimera formation, such as template damage, denaturation time before and during thermocycling, polymerase extension time, and reaction volume. We found two related factors that contribute to chimera formation: the amount of input template into the PCR reaction, and the number of PCR cycles.A second problem that can confound analysis is sequence errors generated during amplification and sequencing. With the updated algorithm for circular consensus sequencing (CCS2), single-molecule reads can be filtered to 99.99% predicted accuracy. Substitution errors in these highly filtered reads may be dominated by mis-incorporations during amplification. Sequence differences in full-length 16S amplicons from several commercial high-fidelity PCR kits were compared.We show results of our experiments and describe our optimized protocol for full-length 16S amplification for SMRT Sequencing. These optimizations have broader implications for other applications that use PCR amplification to phase variations across targeted regions and generate highly accurate reference sequences.

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June 1, 2021  |  

An improved circular consensus algorithm with an application to detect HIV-1 Drug Resistance Associated Mutations (DRAMs)

Scientists who require confident resolution of heterogeneous populations across complex regions have been unable to transition to short-read sequencing methods. They continue to depend on Sanger sequencing despite its cost and time inefficiencies. Here we present a new redesigned algorithm that allows the generation of circular consensus sequences (CCS) from individual SMRT Sequencing reads. With this new algorithm, dubbed CCS2, it is possible to reach high quality across longer insert lengths at a lower cost and higher throughput than Sanger sequencing. We applied CCS2 to the characterization of the HIV-1 K103N drug-resistance associated mutation in both clonal and patient samples. This particular DRAM has previously proved to be clinically relevant, but challenging to characterize due to regional sequence context. First, a mutation was introduced into the 3rd position of amino acid position 103 (A>C substitution) of the RT gene on a pNL4-3 backbone by site-directed mutagenesis. Regions spanning ~1.3 kb were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. Additionally, the proviral reservoir of a subject with known dates of virologic failure of an Efavirenz-based regimen and with documented emergence of drug resistant (K103N) viremia was sequenced at several time points as a proof-of-concept study to determine the kinetics of retention and decay of K103N.Sequencing data were analyzed using the new CCS2 algorithm, which uses a fully-generative probabilistic model of our SMRT Sequencing process to polish consensus sequences to high accuracy. With CCS2, we are able to achieve a per-read empirical quality of QV30 (99.9% accuracy) at 19X coverage. A total of ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40 (99.99%) were obtained for each sample. We demonstrate a 0% miscall rate in both unmixed control samples, and estimate a 48:52 frequency for the K103N mutation in the mixed (50:50) plasmid sample, consistent with data produced by orthogonal platforms. Additionally, the K103N escape variant was only detected in proviral samples from time points subsequent (19%) to the emergence of drug resistant viremia. This tool might be used to monitor the HIV reservoir for stable evolutionary changes throughout infection.

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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.

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June 1, 2021  |  

Multiplexing strategies for microbial whole genome SMRT Sequencing

As the throughput of the PacBio Systems continues to increase, so has the desire to fully utilize SMRT Cell sequencing capacity to multiplex microbes for whole genome sequencing. Multiplexing is readily achieved by incorporating a unique barcode for each microbe into the SMRTbell adapters and using a streamlined library preparation process. Incorporating barcodes without PCR amplification prevents the loss of epigenetic information and the generation of chimeric sequences, while eliminating the need to generate separate SMRTbell libraries. We multiplexed the genomes of up to 8 unique strains of H. pylori. Each genome was sheared and processed through adapter ligation in a single, addition-only reaction. The barcoded samples were pooled in equimolar quantities and a single SMRTbell library was prepared. We demonstrate successful de novo microbial assembly from all multiplexes tested (2- through 8-plex) using data generated from a single SMRTbell library, run on a single SMRT Cell with the PacBio RS II, and analyzed with standard SMRT Analysis assembly methods. This strategy was successful using both small (1.6 Mb, H. pylori) and medium (5 Mb, E. coli) genomes. This protocol facilitates the sequencing of multiple microbial genomes in a single run, greatly increasing throughput and reducing costs per genome.

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June 1, 2021  |  

Complete telomere-to-telomere de novo assembly of the Plasmodium falciparum genome using long-read sequencing

Sequence-based estimation of genetic diversity of Plasmodium falciparum, the most lethal malarial parasite, has proved challenging due to a lack of a complete genomic assembly. The skewed AT-richness (~80.6% (A+T)) of its genome and the lack of technology to assemble highly polymorphic sub-telomeric regions that contain clonally variant, multigene virulence families (i.e. var and rifin) have confounded attempts using short-read NGS technologies. Using single molecule, real-time (SMRT) sequencing, we successfully compiled all 14 nuclear chromosomes of the P. falciparum genome from telomere-to-telomere in single contigs. Specifically, amplification-free sequencing generated reads of average length 12 kb, with =50% of the reads between 15.5 and 50 kb in length. A hierarchical genome assembly process (HGAP), was used to assemble the P. falciparum genome de novo. This assembly accurately resolved centromeres (~90-99% (A+T)) and sub-telomeric regions, and identified large insertions and duplications in the genome that added extra genes to the var and rifin virulence families, along with smaller structural variants such as homopolymer tract expansions. These regions can be used as markers for genetic diversity during comparative genome analyses. Moreover, identifying the polymorphic and repetitive sub-telomeric sequences of parasite populations from endemic areas might inform the link between structural variation and phenotypes such as virulence, drug resistance and disease transmission.

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June 1, 2021  |  

WGS SMRT Sequencing of patient samples from a fecal microbiota transplant trial

Fecal samples were obtained from human subjects in the first blinded, placebo-controlled trial to evaluate the efficacy and safety of fecal microbiota transplant (FMT) for treatment of recurrent C. difficile infection. Samples included pre-and post-FMT transplant, post-placebo transplant, and the donor control; samples were taken at 2 and 8 week post-FMT. Sequencing was done on the PacBio Sequel System, with the goal of obtaining high quality sequences covering whole genes or gene clusters, which will be used to better understand the relationship between the composition and functional capabilities of intestinal microbiomes and patient health. Methods: Samples were randomly sheared to 2-3 kb fragments, a sufficient length to cover most genes, and SMRTbell libraries were prepared using standard protocols. Libraries were run on the Sequel System, which has a throughput of hundreds of thousands of reads per SMRT Cell, adequate yield to sample the complex microbiomes of post-transplant and donor samples.Results: Here we characterize samples, describe library prep methods and detail Sequel System operation, including run conditions. Descriptive statistics of data output (primary analysis) are presented, along with SMRT Analysis reports on circular consensus sequence (CCS) reads generated using an updated algorithm (CCS2). Final sequencing yields are filtered at various levels of predicted accuracy from 90% to 99.9%. Previous studies done using the PacBio RS II System demonstrated the ability to profile at the species level, and in some cases the strain level, and provided functional insight. Conclusions: These results demonstrate that the Sequel System is well-suited for characterization of complex microbial communities, with the ability for high-throughput generation of extremely accurate single-molecule sequences, each several kilobases in length. The entire process from shearing and library prep through sequencing and CCS analysis can be completed in less than 48 hours.

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June 1, 2021  |  

Profiling complex communities 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. Scaffolding with reads from a PacBio unsheared library produced a complete genome of 2.4 Mb. These results illustrate ways in which long accurate reads benefit analysis of complex communities.

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June 1, 2021  |  

Multiplexing strategies for microbial whole genome sequencing using the Sequel System

For microbial sequencing on the PacBio Sequel System, the current yield per SMRT Cell is in excess relative to project requirements. Multiplexing offers a viable solution; greatly increasing throughput, efficiency, and reducing costs per genome. This approach is achieved by incorporating a unique barcode for each microbial sample into the SMRTbell adapters and using a streamlined library preparation process. To demonstrate performance,12 unique barcodes assigned to B. subtilis and sequenced on a single SMRT Cell. To further demonstrate the applicability of this method, we multiplexed the genomes of 16 strains of H. pylori. Each DNA was sheared to 10 kb, end-repaired and ligated with a barcoded adapter in a single-tube reaction. The barcoded samples were pooled in equimolar quantities and a single SMRTbell library was prepared. Successful de novo microbial assemblies were achieved from all multiplexes tested (12-, and 16-plex) using data generated from a single SMRTbell library, run on a single SMRT Cell 1M with the PacBio Sequel System, and analyzed with standard SMRT Analysis assembly methods. Here, we describe a protocol that facilitated the multiplexing up to 12-plex of microbial genomes in one SMRT Cell 1M on the Sequel System that produced near-complete microbial de novo assemblies of <10 contigs for genomes <5 Mb in size.

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June 1, 2021  |  

Multiplexed complete microbial genomes on the Sequel System

Microbes play an important role in nearly every part of our world, as they affect human health, our environment, agriculture, and aid in waste management. Complete closed genome sequences, which have become the gold standard with PacBio long-read sequencing, can be key to understanding microbial functional characteristics. However, input requirements, consumables costs, and the labor required to prepare and sequence a microbial genome have in the past put PacBio sequencing out of reach for some larger projects. We have developed a multiplexed library prep approach that is simple, fast, and cost-effective, and can produce 4 to 16 closed bacterial genomes from one Sequel SMRT Cell. Additionally, we are introducing a streamlined analysis pipeline for processing multiplexed genome sequence data through de novo HGAP assembly, making the entire process easy for lab personnel to perform. Here we present the entire workflow from shearing through assembly, with times for each step. We show HGAP assembly results with single or very few contigs from bacteria from different size genomes, sequenced without or with size selection. These data illustrate the benefits and potential of the PacBio multiplexed library prep and the Sequel System for sequencing large numbers of microbial genomes.

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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.

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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.

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June 1, 2021  |  

Comparative metagenome-assembled genome analysis of “Candidatus Lachnocurva vaginae”, formerly known as Bacterial Vaginosis Associated bacterium – 1 (BVAB1)

Bacterial Vaginosis Associated bacterium 1 (BVAB1) is an as-yet uncultured bacterial species found in the human vagina that belongs to the family Lachnospiraceae within the order Clostridiales. As its name suggests, this bacterium is often associated with bacterial vaginosis (BV), a common vaginal disorder that has been shown to increase a woman’s risk for HIV, Chlamydia trachomatis, and Neisseria gonorrhoeae infections as well as preterm birth. Further, BVAB1 is associated with the persistence of BV following metronidazole treatment, increased vaginal inflammation, and adverse obstetrics outcomes. There is no available complete genome sequence of BVAB1, which has made it di?cult to mechanistically understand its role in disease. We present here a circularized metagenome-assembled genome (cMAG) of B VAB1 as well as a comparative analysis including an additional six metagenome-assembled genomes (MAGs) of this species. These sequences were derived from cervicovaginal samples of seven separate women. The cMAG is 1.649 Mb in size and encodes 1,578 genes. We propose to rename BVAB1 to “Candidatus Lachnocurva vaginae” based on phylogenetic analyses, and provide genomic evidence that this candidate species may metabolize D-lactate, produce trimethylamine (one of the chemicals responsible for BV-associated odor), and be motile. The cMAG and the six MAGs are valuable resources that will further contribute to our understanding of the heterogeneous etiology of bacterial vaginosis.

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June 1, 2021  |  

Improving long-read assembly of microbial genomes and plasmids

Complete, high-quality microbial genomes are very valuable across a broad array of fields, from environmental studies, to human microbiome health, food pathogen surveillance, etc. Long-read sequencing enables accurate resolution of complex microbial genomes and is becoming the new standard. Here we report our novel Microbial Assembly pipeline to facilitate rapid, large-scale analysis of microbial genomes. We sequenced a 48-plex library with one SMRT Cell 8M on the Sequel II System, demultiplexed, then analyzed the data with Microbial Assembly.

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