North American User Group Meeting: Highlights in Human, Animal, Insect, and Microbes

Many thanks to all the PacBio users who attended our annual user group meeting, hosted in St. Louis for the first time. It was great to see so many people sharing best practices and project ideas. If you couldn’t attend, this recap will give you a sense of the highlights from the two day meeting on Wash U’s campus and exciting networking event at the City Museum. You can also download several of the presentations and view video recordings.
Tina Graves-Lindsay from the McDonnell Genome Institute and the Genome Reference Consortium spoke about the importance of phasing human reference genomes. Her team is now working on its fifteenth human genome assembly — part of a major effort to improve genomic representation of ethnic diversity — with a pipeline that generates 60-fold PacBio coverage for a de novo assembly, followed by scaffolding with 10x Genomics or Bionano Genomics technology. They are also using FALCON-Unzip to separate haplotypes, leading to reference-grade diploid assemblies. This approach has already helped resolve errors seen in other genomes and even the gold-standard GRCh38 build of the human reference genome.
Human disease studies were the focus for two of our plenary speakers. Thomas Ray from Duke University spoke about the molecular foundation for wiring the nervous system and studies incorporating the Iso-Seq method to generate an accurate catalog of isoforms — including some that had never been seen before — that may explain how a given gene is able to control distinct neurodevelopmental functions. The project he shared centered on retinal development, covering dystrophies and other conditions that can cause blindness.
Nenad Svrzikapa from Wave Life Sciences spoke about the importance of being able to determine the haplotype of long reads and the application of SMRT Sequencing in Wave’s PRECISION-HD1 and PRECISION-HD2 clinical trials for the treatment of Huntington’s disease.  Huntington’s disease (HD) is a devastating autosomal dominant disorder characterized by cognitive decline, psychiatric illness and chorea.  HD patients carry both a disease-causing mutant allele (mHTT) of the huntingtin gene (HTT) with an aberrant CAG repeat expansion and a functional wild-type allele (wtHTT).  Svrzikapa highlighted that Wave’s allele selective technology enables the specific targeting of the mHTT mRNA by targeting two single nucleotide polymorphisms (SNPs) distal to the CAG repeat region.  Wave used SMRT technology to bridge this distance and developed an investigational assay for phasing the SNPs with the CAG repeat.  Svrzikapa showed the results of Wave’s observational study, which is the first to prospectively identify the frequency of these SNPs in patients with HD, opening the possibility that these patients may be candidates for SNP-targeted therapies such as those being developed by Wave in the PRECISION-HD1 and PRECISION-HD2 clinical trials.
Moving to the animal world, Tim Smith (@tplsmith) from the USDA’s Agricultural Research Service spoke about efforts to generate reference-grade genome assemblies for various bovine species and analyze them to understand factors such as how selective breeding has affected certain breeds. Genome assemblies he cited spanned cattle, water buffalo, and gaur. Smith showed data for each assembly, noting that as data production shifted to the Sequel System, long-read PacBio data became even better at producing highly contiguous assemblies. He shared that one of the most recent, of the Yaklander cattle interspecies, is now the best bovine assembly ever produced. Smith attributed some of the assembly quality to help from the NHGRI’s Sergey Koren (@sergekoren), who also spoke at the meeting. Koren shared his TrioBinning tool, which is useful for resolving haplotypes in virtually any species. It works well even at low coverage but does require a trio of genomes for analysis.
Insects were represented as well in talks from Evgeny Zakharov of the Canadian Centre for DNA Barcoding and Marcé Lorenzen at North Carolina State University. Zakharov focused on the analysis of vertebrate-feeding arthropods to shed light on a region’s broader biodiversity. These complex samples can be interpreted by sequencing biological barcodes, work for which he and his team implemented the Sequel System two years ago to replace Sanger sequencing. Result concordance between the two technologies was excellent, but SMRT Sequencing is much higher-throughput and lower-cost, Zakharov said. That’s why he plans to use this platform for the next phase of this study, which will involve looking at 1.5 million species. In a lightning talk, Lorenzen talked about finding upstream promoters in non-model insects, a project for which she’s using low-coverage SMRT Sequencing. This work is aimed at using molecular genetics to make these pesks “less pesky,” she told attendees.
Microbial genomes were also on display at the user group meeting. Garth Ehrlich from Drexel University spoke about developing a microbiome assay that uses SMRT Sequencing to provide high-quality coverage of the 16S bacterial rRNA for species identification. The goal is a test that would enable de novo identification, no a priori knowledge required. Ehrlich showed how well his assay performs in a number of mock microbial samples, as well as in a case-control study of samples from lung cancer patients and healthy people.
Two lightning talks also fell into the microbial category. Jonathan Jacobs (@jmjacobs2) from The Ohio State University focused on plant pathogenic bacteria, which are characterized by difficult genomic repeats. Elucidating these transcription activator-like effectors can offer clues about virulence and DNA binding patterns, but long-read PacBio sequencing is needed to resolve the differences in these repeats. Microbial multiplexing makes the process quick and affordable. In the other lightning talk, Ben Auch (@sciberius) from the University of Minnesota, a PacBio certified service provider, also spoke about microbial multiplexing, which his lab performs to analyze both Gram-positive and Gram-negative species. His approach generates about 9 Gb per SMRT Cell and routinely produces single-contig assemblies at a total cost of just $250 for a microbe with a genome of 5 Mb.
Two other lightning talk speakers offered application-driven presentations on the Iso-Seq method and amplicon sequencing. Katy Munson (@zhaneel779) from the University of Washington offered pro tips for providing the Iso-Seq method as a service, walking through her recommendations for minimum RNA requirements (5 ug), RIN scores (7.5 or better), and other best practices. Her presentation also showed helpful examples of results from degraded samples. In a separate talk, Dave Corney from GENEWIZ, a PacBio certified service provider, focused on the use of PacBio short and long amplicon sequencing for diverse applications. With the quality of SMRT Sequencing, he predicted that it would be the ability to amplify an amplicon that would become the bottleneck, rather than the sequencing part of the workflow. He also said that high single-molecule fidelity would pave the way for novel applications and ultimately even clinical diagnostics.
The user group meeting concluded with a presentation from our CSO Jonas Korlach about future developments in SMRT Sequencing. He spoke about the upcoming Sequel System 6.0 release of new chemistry, SMRT Cells, and analysis tools that will improve phased de novo assemblies, overall accuracy, and variant detection. He also showed early data from a prototype of the SMRT Cell 8M, which has 8-times the capacity of our current SMRT Cell 1M and is scheduled to be released in 2019.
We’d like to thank our hosts for the meeting, the McDonnell Genome Institute at Washington University School of Medicine, as well as our partners: Advanced Analytical Technologies, Diagenode, DNAnexus, Integrated DNA Technologies, PerkinElmer, Phase Genomics, and Sage Science.