June 1, 2021  |  

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens.

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic states. Therefore, sequencing methods which provide complete, de novo genome assemblies and epigenomes are necessary to fully characterize infectious disease agents in an unbiased, hypothesis-free manner. Hybrid assembly methods have been described that combine long sequence reads from SMRT DNA Sequencing with short reads (SMRT CCS (circular consensus) or second-generation reads), wherein the short reads are used to error-correct the long reads which are then used for assembly. We have developed a new paradigm for microbial de novo assemblies in which SMRT sequencing reads from a single long insert library are used exclusively to close the genome through a hierarchical genome assembly process, thereby obviating the need for a second sample preparation, sequencing run, and data set. We have applied this method to achieve closed de novo genomes with accuracies exceeding QV50 (>99.999%) for numerous disease outbreak samples, including E. coli, Salmonella, Campylobacter, Listeria, Neisseria, and H. pylori. The kinetic information from the same SMRT Sequencing reads is utilized to determine epigenomes. Approximately 70% of all methyltransferase specificities we have determined to date represent previously unknown bacterial epigenetic signatures. With relatively short sequencing run times and automated analysis pipelines, it is possible to go from an unknown DNA sample to its complete de novo genome and epigenome in about a day.


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  |  

An improved circular consensus algorithm with an application to detection of 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 arbitrarily high quality across longer insert lengths at a lower cost and higher throughput than Sanger Sequencing. We apply this new algorithm, dubbed CCS2, to the characterization of the HIV-1 K103N drug-resistance associated mutation, which is both important clinically, and represents a challenge due to regional sequence context. 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 ~1,300 bp were PCR amplified from both the non-mutated and mutant (K103N) plasmids, and were sequenced individually and as a 50:50 mixture. 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 arbitrarily high accuracy. This result, previously demonstrated for multi-molecule consensus sequences with the Quiver algorithm, is made possible by incorporating per-Zero Mode Waveguide (ZMW) characteristics, thus accounting for the intrinsic changes in the sequencing process that are unique to each ZMW. With CCS2, we are able to achieve a per-read empirical quality of QV30 with 19X coverage. This yields ~5000 1.3 kb consensus sequences with a collective empirical quality of ~QV40. Additionally, we demonstrate a 0% miscall rate in both unmixed samples, and estimate a 48:52% frequency for the K103N mutation in the mixed sample, consistent with data produced by orthogonal platforms.


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.


June 1, 2021  |  

T-cell receptor profiling using PacBio sequencing of SMARTer libraries

T-cells play a central part in the immune response in humans and related species. T-cell receptors (TCRs), heterodimers located on the T-cell surface, specifically bind foreign antigens displayed on the MHC complex of antigen-presenting cells. The wide spectrum of potential antigens is addressed by the diversity of TCRs created by V(D)J recombination. Profiling this repertoire of TCRs could be useful from, but not limited to, diagnosis, monitoring response to treatments, and examining T-cell development and diversification.


June 1, 2021  |  

Best practices for diploid assembly of complex genomes using PacBio: A case study of Cascade Hops

A high quality reference genome is an essential resource for plant and animal breeding and functional and evolutionary studies. The common hop (Humulus lupulus, Cannabaceae) is an economically important crop plant used to flavor and preserve beer. Its genome is large (flow cytometrybased estimates of diploid length >5.4Gb1), highly repetitive, and individual plants display high levels of heterozygosity, which make assembly of an accurate and contiguous reference genome challenging with conventional short-read methods. We present a contig assembly of Cascade Hops using PacBio long reads and the diploid genome assembler, FALCON-Unzip2. The assembly has dramatically improved contiguity and completeness over earlier short-read assemblies. The genome is primarily assembled as haplotypes due to the outbred nature of the organism. We explore patterns of haplotype divergence across the assembly and present strategies to deduplicate haplotypes prior to scaffolding


June 1, 2021  |  

No-amp targeted SMRT sequencing using a CRISPR-Cas9 enrichment method

Targeted sequencing of genomic DNA requires an enrichment method to generate detectable amounts of sequencing products. Genomic regions with extreme composition bias and repetitive sequences can pose a significant enrichment challenge. Many genetic diseases caused by repeat element expansions are representative of these challenging enrichment targets. PCR amplification, used either alone or in combination with a hybridization capture method, is a common approach for target enrichment. While PCR amplification can be used successfully with genomic regions of moderate to high complexity, it is the low-complexity regions and regions containing repetitive elements sometimes of indeterminate lengths due to repeat expansions that can lead to poor or failed PCR enrichment. We have developed an enrichment method for targeted SMRT Sequencing on the PacBio Sequel System using the CRISPR-Cas9 system that requires no PCR amplification. Briefly, a preformed SMRTbell library containing the target region of interest is cleaved with Cas9 through direct interaction with a sequence-specific guide RNA. After ligation with new poly(A) hairpin adapters, the asymmetric SMRTbell templates are enriched by magnetic bead separation. This method, paired with SMRT Sequencing’s long reads, high consensus accuracy, and uniform coverage, allows sequencing of genomic regions regardless of challenging sequence context that cannot be investigated with other technologies. The method is amenable to analyzing multiple samples and/or targets in a single reaction. In addition, this method also preserves epigenetic modifications allowing for the detection and characterization of DNA methylation which has been shown to be a key factor in the disease mechanism for some repeat expansion diseases. Here we present results of our latest No-Amp Targeted Sequencing procedure applied to the characterization of CAG triplet repeat expansions in the HTT gene responsible for Huntington’s Disease.


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