Scientific posters

The SARS-CoV-2 global pandemic has highlighted the utility of pathogen surveillance pipelines that provide comprehensive genomic information, giving public health scientists a more complete view of the spread and characteristics of circulating pathogens. Beyond COVID-19, there is great interest in public health to expand high resolution surveillance to other infectious diseases. Highly accurate, long HiFi reads produced by the PacBio Sequel IIe System have brought new levels of contiguity, completeness, and accuracy to large genome assembly. HiFi reads are similarly beneficial for microbial genome assembly, as higher quality assemblies enhance our ability to investigate foodborne illnesses and monitor antimicrobial resistance. However, obstacles in library preparation workflow, cost, and recovery of small plasmids have limited use in public health. Here, we introduce a new library prep workflow and assembly algorithm based on HiFi reads that enables a high throughput, end-to-end solution for microbial genome assembly. The new workflow combines steps, eliminates the need for strict size selection, shortens the total time to 6 hours, and enables library prep automation. The assembly algorithm uses strict read-to-read overlaps enabled by HiFi read accuracy to resolve repeats. It uses a two-stage approach to first assemble chromosomes and then recover short, high-coverage plasmids. To evaluate the method, a pool of HiFi libraries with 96 microbial samples and total genome size of 375 Mb was generated. The protocol was evaluated with microbes relevant to pathogen surveillance including common foodborne pathogens (Listeria, Salmonella) and species often seen in hospital settings (Klebsiella, Staphylococcus). The microbes represent a range of genome sizes, assembly complexity, GC content, chromosome counts, and plasmid content. DNA samples were sheared to 7 kb - 10 kb, prepared as barcoded libraries, pooled, and sequenced on one SMRT Cell 8M on the Sequel IIe System. Reference quality de novo microbial assemblies with 5 contigs or fewer were achieved for all samples. Typical chromosome assembly quality was Q50, measured as concordance to reference assemblies. Nearly all plasmids were recovered, including those shorter than 5 kb which are often lost in workflows with strict size selection. Taken together, the new method provides a high-throughput, cost-effective approach suitable to routinely generate reference quality microbial genomes in a public health environment as part of a pathogen surveillance program.

The COVID-19 pandemic is an ongoing global challenge, with the repeated emergence of new variants that are more contagious, more virulent, drug resistant or evade vaccine-induced immunity. In response, the HiFiViral SARS-CoV-2 kit was developed as a scalable solution with increased resilience against virus mutations, designed for use on the Sequel IIe system. Unlike PCR-based amplicon methods, the HiFiViral SARS-CoV-2 kit relies on ~1,000, densely tiled Molecular Inversion Probes (MIPs) such that every genomic position is covered by ~22 probes, resulting in robust genome coverage of all circulating variants, including the mutation-dense Omicron lineage, across a broad range of Ct values. The HiFiViral kit offers many benefits for high-throughput surveillance. Sequencing 675 bp fragments with highly accurate HiFi reads enables comprehensive variant detection, including single nucleotide variants, indels, structural variants, and identification of multi-strain samples. The kit is scalable, containing all reagents needed to enrich and barcode 384 samples, in batches ranging from 24 – 384, for sequencing in one SMRT bell library. For high throughput labs, up to 8 SMRT Cells may be loaded on an instrument with no subsequent touch points, for up to 3,072 samples per run. The enrichment assay is also simpler to execute than PCR-based assays, consisting of just 4 add-only, color-change indicated steps. Barcoding primers come pre-mixed in a 384-well, resealable plate. The simple assay design uses fewer plastics, limiting the impact of supply chain issues. In addition, methods for running the HiFiViral kit were established on a mosquito® HV Genomics pipetting robot and assessed for their performance. Finally, SMRT Link data analysis is one touch, and reports include variant calling, genome coverage, multi-strain detection, and a plate performance summary. File outputs include consensus sequences ready for database submission and reads and consensus sequences aligned to the Wuhan reference. In this study, we demonstrate consistent recovery of >95% complete SARS-CoV-2 genomes using the commercially available HiFiViral kit at 4 different sites performing routine genomic surveillance. The evaluation runs included a broad range of sample Ct inputs across control and nasopharyngeal samples in batches up to 384. The runs also demonstrated consistent performance against Alpha, Delta, Omicron and other variant lineages, without the need for probe updates. In summary, the HiFiViral for SARS-CoV-2 kit is a cost-effective, convenient, accurate method for viral sequencing and well-suited for scalable surveillance of a rapidly evolving virus to inform public health decision making. ¹PacBio, 1305 O'Brien Drive, Menlo Park, CA 94025 ²SPT Labtech, Ltd., Melbourn Science Park ,Melbourn, Hertfordshire SG8 6HB

We developed a novel method to comprehensively interrogate a panel of 43 pharmacogenomic genes using an enrichment-based capture strategy (IDT) coupled with PacBio highly accurate (HiFi) Sequencing.

The COVID-19 pandemic continues to be a major global epidemiological challenge with the ongoing emergence of new strain lineages that are more contagious, more virulent, drug resistant and in some cases evade vaccine-induced immunity. In response, the HiFiViral SARS-CoV-2 kit (PacBio; Menlo Park, California) was developed as a scalable solution for the Sequel II and Sequel IIe systems. The HiFiViral SARS-CoV-2 is an easy to perform solution for surveillance of variants to support pandemic response by public health. With 80% of samples yielding complete genome coverage in a 96-plex run, the combination of long read lengths and a differentiated probe design provides highly accurate results and robust genome coverage across a range of Ct values.

With PacBio Single Molecule, Real-Time (SMRT) Sequencing on the Sequel IIe System you can characterize highly polymorphic CYP2D6 locus from 384 or more samples with just one SMRT Cell. Explore our applications and pricing to get your sequencing project started.

Many genetic diseases are mapped to structurally complex loci. These regions contain highly similar paralogous alleles (>99% identity) that span kilobases within the human genome. Comprehensive screening for pathogenic variants is incomplete and labor intensive using short-reads or optical mapping. In contrast, long-range amplification and PacBio HiFi sequencing fully and directly resolve and phase a wide range of pathogenic variants without inference. To capitalize on the accuracy of HiFi data we designed a new amplicon analysis tool, pbAA. pbAA can rapidly deconvolve a mixture of haplotypes, enabling precise diplotyping, and disease allele classification.

Targeted amplification of difficult pharmacogenetic loci with PacBio HiFi reads can resolve complex alleles in a single direct assay without imputation.

New workflow for preparing SMRTbell libraries from <4ug gDNA input using AMPure PB bead purification. This workflow requires samples with high molecular weight gDNA with minimal presence of <5kb fragments.

There are many clinically important genes in “dark” regions of the human genome. These regions are characterized as dark due to a paucity of NGS coverage as a result of short-read sequencing or mapping difficulties. Low NGS sequencing yield can arise in these regions due to the presence of various repeat elements or biased base composition while inaccurate mapping can result from segmental duplications. Long-read sequencing coupled with an optimized, robust enrichment method has the potential to illuminate these dark regions.

Using concatenation we increase single-cell Iso-Seq (scIso-Seq) throughput to ~8 million de-concatenated full-length molecules per SMRT Cell 8M. We show that the scIso-Seq method captures full-length isoform information at the single-cell level.

The highly polymorphic CYP2D6 gene impacts the metabolism of 25% of the mostly prescribed drugs. Thus, accurate identification of variant CYP2D6 alleles in individuals is necessary for personalized medicine. PacBio HiFi sequencing produces long and accurate reads to identify variant regions. Here, we describe an end-to-end workflow for the characterization of full-length CYP2D6 by HiFi sequencing.

Many genetic diseases are mapped to structurally complex loci. These regions contain highly similar paralogous alleles (>99% identity) that span kilobases within the human genome. Comprehensive screening for pathogenic variants is incomplete and labor intensive using short-reads or optical mapping. In contrast, long-range amplification and PacBio HiFi sequencing fully and directly resolve and phase a wide range of pathogenic variants without inference. To capitalize on the accuracy of HiFi data we designed a new amplicon analysis tool, pbAA. pbAA can rapidly deconvolve a mixture of haplotypes, enabling precise diplotyping, and disease allele classification.

There are many clinically important genes in “dark” regions of the human genome. These regions are characterized as dark due to a paucity of NGS coverage as a result of short-read sequencing or mapping difficulties. Low NGS sequencing yield can arise in these regions due to the presence of various repeat elements or biased base composition while inaccurate mapping can result from segmental duplications. Long-read sequencing coupled with an optimized, robust enrichment method has the potential to illuminate these dark regions.

CYP2D6 is a highly polymorphic gene with more than 130 named variants, including deletions, duplications, single nucleotide polymorphisms, and other types of variation (Butler, 2018; Black et al., 2011). These variants affect the rate of metabolism in human individuals of approximately 25% of common prescription drugs (Owen et al., 2019;). PacBio SMRT sequencing is a proven tool for the interrogation of CYP2D6 variants (Qiao et al., 2016; Buermans et al., 2017).  Now with HiFi sequencing, we have developed a streamlined end-to-end workflow for the more accurate detection of highly polymorphic CYP2D6 loci. This study also evaluates the advantage of HiFi reads for the sequencing of full-length CYP2D6 genes with variants previously annotated by other technologies. Twenty-two Coriell pharmacogenomic samples containing variant CYP2D6 alleles were amplified using long-range PCR. The primer pairs for the amplification of upstream CYP2D6 gene duplications and the downstream CYP2D6 genes were adapted from a publication in Pharmacogenomics (Qiao et al., 2019). A 2-step PCR method was used for the addition of the unique barcode to each sample, allowing pooling of multiple samples for SMRTbell library prep. The resulting SMRTbell Library was then sequenced on the PacBio Sequel II/IIe system for 20-hours. HiFi reads (>QV20) were demultiplexed on SMRTlink and clustered into haplotypes. The consensus reads of each haplotype were produced using the “pbaa” amplicon analysis and then mapped to the human reference genome GRCh38 for the assignment of CYP2D6 types. More than 700,000 full-length HiFi reads were generated with an average read length of 8.2 kb and a mean accuracy of 99.9%. Nearly all (>99%) demultiplexed reads were on target to the CYP2D6 locus. Genotyping of the CYP2D6 region with PacBio HiFi reads identified all expected upstream duplications and downstream CYP2D6 alleles including single nucleotide variants, except for *5 allele which is a complete deletion. For 21 of 22 samples, the types from HiFi reads matched the diplotypes identified from microarrays and qPCR, while providing full resolution of each allele. One sample was identified as being mistyped by microarray as *1/*41. HiFi sequencing produced a correct type of *33/*41. In addition, for 4/21 samples HiFi sequencing identified duplications missed by microarray or real-time PCR. The PCR and sequencing assay we have presented here for the detection of CYP2D6 variants is robust and specific. Assignment of new alleles or duplications on pharmacogenomic samples from HiFi reads suggests that PacBio sequencing technology can reveal new diplotypes that were not characterized accurately by other technologies. This study demonstrates that HiFi sequencing provides much higher resolution than either microarray or real-time PCR for the detection of polymorphic genes, while maintaining sensitivity and accuracy.

Many genetic diseases are mapped to structurally complex loci. These regions contain highly similar paralogous alleles (>99% identity) that span kilobases within the human genome. Comprehensive screening for pathogenic variants amongst paralogous sequences is incomplete and labor intensive using short-reads or optical mapping. In contrast, long-range targeted amplification and PacBio HiFi sequencing fully and directly resolves and phases a wide range of pathogenic variants without assembly or inference. To capitalize on the accuracy of HiFi amplicon data we designed a new amplicon analysis tool, pbAA. pbAA uses a new sequence clustering algorithm to rapidly deconvolve (separate) a mixture of haplotypes, enabling precise diplotyping, and disease allele classification. In this experiment, we analyzed two sets of gene-pseudogene systems, GBA and CYP, that are the second and eighth most common carrier disease alleles, respectively. Samples tested were selected from the Coriell catalog known to have pathogenic variants troublesome to test for with standard short-read assays. Co-amplified long-range PCR amplicons were generated for GBA (12kb)/GBAP1 (15kb), responsible for Gaucher disease, as well as CYP21A2 (10kb)/CYP21A1P (8kb), responsible for congenital adrenal hyperplasia. We obtained 7 samples to test the CYP21A2 region and 13 separate samples for GBA. HiFi reads were then generated from the amplicon libraries on both Sequel and Sequel II Systems, with replicated samples, to achieve a 24-sample multiplex for each target. Consensus amplicons were produced using pbAA and variants were determined using minimap2 alignments along with a custom SQL database for characterizing and reporting results. From these data we were able to accurately call all pathogenic variants in the test samples for all replicates, including whole-gene deletions, gene duplication, gene fusions, recombinant exons, and phased complex heterozygotes. In one trio affected by adrenal hyperplasia, three large structural variants were correctly and independently attributed to the parents and proband, including a duplication of CP21A1P and a CYP21A1P-CYP21A2 gene fusion in the mother and a CYP21A2 deletion in the father. This experiment demonstrates how PacBio HiFi data, analyzed with pbAA, simplifies targeted disease allele identification.