Menu
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. 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 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 the 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% accuracy can be obtained from Circular Consensus Sequencing.


June 1, 2021  |  

Analysis of full-length metagenomic 16S genes by Single Molecule, Real-Time Sequencing

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.


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  |  

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  |  

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. 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 a mixture of sequences with close similarities, the products may contain chimeras, or recombinant molecules, at rates as high as 20-30%. These PCR artifacts make it difficult to identify novel species, and reduce the amount of productive sequences. We investigated multiple factors that have been hypothesized to contribute to chimera formation, such as template damage, denaturing time before and during cycling, polymerase extension time, and reaction volume. Of the factors tested, we found two major related contributors to chimera formation: the amount of input template into the PCR reaction and the number of PCR cycles. Sequence errors generated during amplification and sequencing can also confound the analysis of complex populations. Circular Consensus Sequencing (CCS) can generate single-molecule reads with >99% accuracy, and the SMRT Analysis software provides filtering of these reads to >99.99% accuracies. Remaining substitution errors in these highly-filtered reads are likely dominated by mis-incorporations during amplification. Therefore, we compared the impact of several commercially-available high-fidelity PCR kits with full-length 16S amplification. We show results of our experiments and describe an 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 to generate highly accurate reference sequences.


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.


April 21, 2020  |  

Evolution and global transmission of a multidrug-resistant, community-associated MRSA lineage from the Indian subcontinent

The evolution and global transmission of antimicrobial resistance has been well documented in Gram-negative bacteria and healthcare-associated epidemic pathogens, often emerging from regions with heavy antimicrobial use. However, the degree to which similar processes occur with Gram-positive bacteria in the community setting is less well understood. Here, we trace the recent origins and global spread of a multidrug resistant, community-associated Staphylococcus aureus lineage from the Indian subcontinent, the Bengal Bay clone (ST772). We generated whole genome sequence data of 340 isolates from 14 countries, including the first isolates from Bangladesh and India, to reconstruct the evolutionary history and genomic epidemiology of the lineage. Our data shows that the clone emerged on the Indian subcontinent in the early 1970s and disseminated rapidly in the 1990s. Short-term outbreaks in community and healthcare settings occurred following intercontinental transmission, typically associated with travel and family contacts on the subcontinent, but ongoing endemic transmission was uncommon. Acquisition of a multidrug resistance integrated plasmid was instrumental in the divergence of a single dominant and globally disseminated clade in the early 1990s. Phenotypic data on biofilm, growth and toxicity point to antimicrobial resistance as the driving force in the evolution of ST772. The Bengal Bay clone therefore combines the multidrug resistance of traditional healthcare-associated clones with the epidemiological transmission of community-associated MRSA. Our study demonstrates the importance of whole genome sequencing for tracking the evolution of emerging and resistant pathogens. It provides a critical framework for ongoing surveillance of the clone on the Indian subcontinent and elsewhere.Importance The Bengal Bay clone (ST772) is a community-acquired and multidrug-resistant Staphylococcus aureus lineage first isolated from Bangladesh and India in 2004. In this study, we show that the Bengal Bay clone emerged from a virulent progenitor circulating on the Indian subcontinent. Its subsequent global transmission was associated with travel or family contact in the region. ST772 progressively acquired specific resistance elements at limited cost to its fitness and continues to be exported globally resulting in small-scale community and healthcare outbreaks. The Bengal Bay clone therefore combines the virulence potential and epidemiology of community-associated clones with the multidrug-resistance of healthcare-associated S. aureus lineages. This study demonstrates the importance of whole genome sequencing for the surveillance of highly antibiotic resistant pathogens, which may emerge in the community setting of regions with poor antibiotic stewardship and rapidly spread into hospitals and communities across the world.


April 21, 2020  |  

Genomic and Functional Analysis of Emerging Virulent and Multidrug-Resistant Escherichia coli Lineage Sequence Type 648.

The pathogenic extended-spectrum-beta-lactamase (ESBL)-producing Escherichia coli lineage ST648 is increasingly reported from multiple origins. Our study of a large and global ST648 collection from various hosts (87 whole-genome sequences) combining core and accessory genomics with functional analyses and in vivo experiments suggests that ST648 is a nascent and generalist lineage, lacking clear phylogeographic and host association signals. By including large numbers of ST131 (n?=?107) and ST10 (n?=?96) strains for comparative genomics and phenotypic analysis, we demonstrate that the combination of multidrug resistance and high-level virulence are the hallmarks of ST648, similar to international high-risk clonal lineage ST131. Specifically, our in silico, in vitro, and in vivo results demonstrate that ST648 is well equipped with biofilm-associated features, while ST131 shows sophisticated signatures indicative of adaption to urinary tract infection, potentially conveying individual ecological niche adaptation. In addition, we used a recently developed NFDS (negative frequency-dependent selection) population model suggesting that ST648 will increase significantly in frequency as a cause of bacteremia within the next few years. Also, ESBL plasmids impacting biofilm formation aided in shaping and maintaining ST648 strains to successfully emerge worldwide across different ecologies. Our study contributes to understanding what factors drive the evolution and spread of emerging international high-risk clonal lineages.Copyright © 2019 American Society for Microbiology.


April 21, 2020  |  

Remodeling of pSK1 Family Plasmids and Enhanced Chlorhexidine Tolerance in a Dominant Hospital Lineage of Methicillin-Resistant Staphylococcus aureus.

Staphylococcus aureus is a significant human pathogen whose evolution and adaptation have been shaped in part by mobile genetic elements (MGEs), facilitating the global spread of extensive antimicrobial resistance. However, our understanding of the evolutionary dynamics surrounding MGEs, in particular, how changes in the structure of multidrug resistance (MDR) plasmids may influence important staphylococcal phenotypes, is incomplete. Here, we undertook a population and functional genomics study of 212 methicillin-resistant S. aureus (MRSA) sequence type 239 (ST239) isolates collected over 32?years to explore the evolution of the pSK1 family of MDR plasmids, illustrating how these plasmids have coevolved with and contributed to the successful adaptation of this persistent MRSA lineage. Using complete genomes and temporal phylogenomics, we reconstructed the evolution of the pSK1 family lineage from its emergence in the late 1970s and found that multiple structural variants have arisen. Plasmid maintenance and stability were linked to IS256- and IS257-mediated chromosomal integration and disruption of the plasmid replication machinery. Overlaying genomic comparisons with phenotypic susceptibility data for gentamicin, trimethoprim, and chlorhexidine, it appeared that pSK1 has contributed to enhanced resistance in ST239 MRSA isolates through two mechanisms: (i) acquisition of plasmid-borne resistance mechanisms increasing the rates of gentamicin resistance and reduced chlorhexidine susceptibility and (ii) changes in the plasmid configuration linked with further enhancement of chlorhexidine tolerance. While the exact mechanism of enhanced tolerance remains elusive, this research has uncovered a potential evolutionary response of ST239 MRSA to biocides, one of which may contribute to the ongoing persistence and adaptation of this lineage within health care institutions. Copyright © 2019 Baines et al.


April 21, 2020  |  

Genomic and transcriptomic characterization of Pseudomonas aeruginosa small colony variants derived from a chronic infection model.

Phenotypic change is a hallmark of bacterial adaptation during chronic infection. In the case of chronic Pseudomonas aeruginosa lung infection in patients with cystic fibrosis, well-characterized phenotypic variants include mucoid and small colony variants (SCVs). It has previously been shown that SCVs can be reproducibly isolated from the murine lung following the establishment of chronic infection with mucoid P. aeruginosa strain NH57388A. Using a combination of single-molecule real-time (PacBio) and Illumina sequencing we identify a large genomic inversion in the SCV through recombination between homologous regions of two rRNA operons and an associated truncation of one of the 16S rRNA genes and suggest this may be the genetic switch for conversion to the SCV phenotype. This phenotypic conversion is associated with large-scale transcriptional changes distributed throughout the genome. This global rewiring of the cellular transcriptomic output results in changes to normally differentially regulated genes that modulate resistance to oxidative stress, central metabolism and virulence. These changes are of clinical relevance because the appearance of SCVs during chronic infection is associated with declining lung function.


April 21, 2020  |  

Evolution of a clade of Acinetobacter baumannii global clone 1, lineage 1 via acquisition of carbapenem- and aminoglycoside-resistance genes and dispersion of ISAba1.

Resistance to carbapenem and aminoglycoside antibiotics is a critical problem in Acinetobacter baumannii, particularly when genes conferring resistance are acquired by multiply or extensively resistant members of successful globally distributed clonal complexes, such as global clone 1 (GC1) . Here, we investigate the evolution of an expanding clade of lineage 1 of the GC1 complex via repeated acquisition of carbapenem- and aminoglycoside-resistance genes. Lineage 1 arose in the late 1970s and the Tn6168/OCL3 clade arose in the late 1990s from an ancestor that had already acquired resistance to third-generation cephalosporins and fluoroquinolones. Between 2000 and 2002, two distinct subclades have emerged, and they are distinguishable via the presence of an integrated phage genome in subclade 1 and AbaR4 (carrying the oxa23 carbapenem-resistance gene in Tn2006) at a specific chromosomal location in subclade 2. Part or all of the original resistance gene cluster in the chromosomally located AbaR3 has been lost from some isolates, but plasmids carrying alternate resistance genes have been gained. In one group in subclade 2, the chromosomally located AbGRI3, carrying the armA aminoglycoside-resistance gene, has been acquired from a GC2 isolate and incorporated via homologous recombination. ISAba1 entered the common ancestor of this clade as part of the cephalosporin-resistance transposon Tn6168 and has dispersed differently in each subclade. Members of subclade 1 share an ISAba1 in one specific position in the chromosome and in subclade 2 two different ISAba1 locations are shared. Further shared ISAba1 locations distinguish further divisions, potentially providing simple markers for epidemiological studies.


Talk with an expert

If you have a question, need to check the status of an order, or are interested in purchasing an instrument, we're here to help.