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This blog features voices from PacBio — and our partners and colleagues — discussing the latest research, publications, and updates about SMRT Sequencing. Check back regularly or sign up to have our blog posts delivered directly to your inbox.

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Tuesday, November 16, 2021

Introducing First Fully-Kitted Solution for SARS-CoV-2 Surveillance and Microbial Whole Genome Assembly with HiFi Sequencing


These developments advance the ability of laboratories to identify and track infectious disease threats

SARS-CoV-2 mutates rapidly but a new solution is now available for public health laboratories in the race against pathogen evolution—the HiFiViral SARS-CoV-2 Kit. Unlike amplicon-based assays that require periodic updates and subsequent revalidation to ensure all known SARS-CoV-2 variants are detectable, the new HiFiViral SARS-CoV-2 Kit gives researchers a more complete view of all mutations, ensuring that emerging variants can be captured for further study.

This fully kitted solution brings trusted, accurate HiFi sequencing to SARS-CoV-2 variant detection. Compared with short read sequencing, HiFi sequencing provides a more comprehensive view of viral variation of all types and enables laboratories to identify multiple viral mutations, including minor variants that may be emerging within each sample.

HiFiViral SARS-CoV-2 KitTo date, amplicon-based technologies for pathogen surveillance have limited the ability of laboratories to identify new SARS-CoV-2 mutations in real time. Some variants affect primer binding, which challenge current Dx detection tests and the ability to call new variants under variant profiling applications. This limitation can hamper efforts to identify new variants and understand their spread across populations. Without surveillance, public health decision makers fly blind to emerging mutations, and this can lengthen the timely response needed to prevent spread.

The HiFiViral SARS-CoV-2 Kit enables cost-effective viral sequencing using molecular inversion probes, which address common challenges of amplicon-based solutions by simplifying workflows, offering mutation resilience, and enabling cost-effective scalability. Paired with SMRT Link v10.2 software, the full solution gives laboratories the ability to perform quality checks on experiments and generate submission-ready files for surveillance. The automated sequencing and analysis workflow can be initiated with one touch and completed overnight.



Microbial Whole Genome Assembly Joins Suite of HiFi WGS Applications

microbesWe are also happy to announce an updated protocol, analysis software, and the SMRTbell Barcoded Adapter Plate 3.0, which bring the power of HiFi reads to microbial whole genome assembly.

The new solution provides:

• 96 barcoded adapters, with improved design for multiplexing

• Increased throughput, doubling the number of samples per run from 48 to 96

• Reduced cost per sample from higher multiplexing

• Fast assembly of reference-quality genomes with the SMRTLink v10.2 Microbial Assembly application for HiFi reads

Public health and research labs now have an improved tool for investigating outbreaks in the food supply, tracking antimicrobial resistance, and fully characterizing emerging pathogen threats. Paired with the new HiFiViral SARS-CoV-2 kit, these innovations equip public health and microbial research labs with a powerful selection of applications to service a broad range of use cases via Sequel II and Sequel IIe systems, setting the standard for high quality reference genomes.

The HiFiViral SARS-CoV-2 kit is currently shipping. The components for the HiFi microbial whole genome assembly application will be available this quarter. Connect with a scientist to learn more how these new products can advance your microbial research.

Read More »

Thursday, November 4, 2021

ASHG 2021: Improving Human Health through Genomics with HiFi Sequencing



The PacBio team recently attended the American Society of Human Genetics (ASHG) 2021 Virtual Meeting. The meeting provided a forum for the presentation and discussion of cutting-edge science in all areas of human genetics. ASHG members and leading scientists from around the world were selected to present their research findings. We were excited to be one of the 80 industry partners who participated in ASHG’s annual meeting.

A highlight of the event was a Fireside Chat with our President and CEO, Christian Henry and President of Omniome, Richard Shen. The two discussed their vision for the combined future of the companies and how PacBio intends to become the first company to offer both long-read and short-read sequencing platforms.

Christian Henry shared his excitement about the recent Omniome acquisition: “Having the ability to focus on the technology and the applications that make the most sense is a unique capability. And then on top of that, considering how we can combine these technologies in unique ways so that customers get more deeply integrated answers is going to be really exciting to see unfold. That is what gets me so excited about this acquisition. We can serve our customers in a new way and no other company can do this.”

Watch the entire discussion:



PacBio participated in multiple sessions throughout the conference by highlighting how HiFi sequencing is helping human health through discoveries in many different fields, such as rare disease, cardiovascular, neurological, and immunological diseases, Alzheimer’s disease, and pharmacogenomics.

Jennifer StoneOur Vice President of Segment Marketing, Jennifer Stone, kicked off our workshop by presenting milestones and seminal publications of PacBio’s technology produced this year, demonstrating how HiFi sequencing changed the game in human genetics. In one specific highlight, she showcased research published in Nature Biotechnology that compares a variety of different sequencing platforms. The authors observed that PacBio had the lowest error rate out of all of the technologies listed – further emphasizing the importance and value of accuracy in sequencing. Watch her full talk, “HiFi Sequencing: See What You’ve Been Missing.”


Emily FarrowEmily Farrow of Children’s Mercy Kansas City highlighted the integrated analyses offered by HiFi sequencing. She noted the increased awareness of the complexity of the human genome and reviewed the challenges of rare disease diagnosis. “Historically, long-read sequencing was difficult to deploy due to higher error rates and lower throughput but as these have been rapidly improved these are now an attractive solution for diagnosing rare disease.” She showcased an example of siblings who had a rare disease and had been seeking insights for 7 years. Dr. Farrow explained the challenges of helping these patients and how long-read technology was able to unravel the mystery of their disease. View her talk, “Integrated Rare Disease Genomics Using Long-Read Genome Sequencing.”


Aziz Al'KhafaliAziz Al’Khafaji (@AzizAlkhafaji), from the Broad Institute, shared a new method called Multiplexed Arrays sequencing (MAS-seq), dramatically increasing the throughput and thereby maximizing the potential of sequencing transcript isoforms on the PacBio platform. He described how short-read sequencing technologies provide only limited data on isoforms and long-read sequencing can span the full length of the vast majority of transcripts enabling direct isoform observations. Watch his presentation, “Scalable RNA Isoform Sequencing using Intramolecular Multiplexed cDNAs.”


Henne HolstegeHenne Holstege (@HolstegeHenne), from the Amsterdam University Medical Center, discussed her work with centenarians and long-read sequencing to identify predominantly expressed VNTRs in the brains of patients who have been diagnosed with Alzheimer’s disease. She shared, “During aging, blood stem cells will acquire more and more somatic mutations and that the blood will become more and more clonal with age. So, one clone will eventually generate more blood cells in the peripheral blood and that is what we will be sequencing. We cannot ignore this aspect of sequencing when we will be sequencing older individuals.” The team found that long genes were more likely to be expressed in the brain and associated with neuropsychological disorders. See her full presentation, “Uncovering Neurological Disorders Through an Examination of VNTRs.”

Beyond the PacBio sponsored events, the research community presented many examples where HiFi sequencing was used to fuel exciting new research. Here are some insights that came to light during the event that emphasize the real-world impacts of long-read sequencing:

Adam Phillippy (@aphillippy), of the T2T consortium, unveiled the first truly complete assembly of the human genome (CHM-13). The long and accurate HiFi reads were instrumental in resolving the missing 8% from Grch38 and the additional content represents >200 Mb of new sequences, 2,226 new genes of which 115 are predicted to be protein coding.

During the Multidisciplinary Omics Approaches for Diagnostic Platform session, two separate talks from Radboud MC and Baylor Genetics addressed the limitations of short-read sequencing to cover areas of high homology due to pseudogenes. Radboud estimated that 3.5% of the coding exome is obscured by pseudogenes. As demonstrated by the T2T consortium, and by many publications, HiFi sequencing has been shown to be very effective in resolving areas of high homology.

Mathew Bainbridge of Rady Children’s Hospital shared a good example of this capability in practice. He presented the identification of a likely pathogenic stop loss variant in the IKBKG gene found by HiFi sequencing in a negative case from a previous attempt with short-read genome sequencing. A pseudogene had obscured this call in the previous attempt with short-read sequencing. In his talk he highlighted the value of HiFi sequencing in resolving clinical genes in “dark regions” and the benefits of phasing, structural variation detection and methylation detection. The goal at Rady’s is to eventually develop a single germline genetic assay that will encompass most of the functionality of today’s individual assays.

The conference presentations provided a firsthand look at how PacBio’s technology has helped further research around the world. The team is proud to be a part of such important work and thanks all of the speakers for sharing their research. We look forward to seeing you all at ASHG 2022 next year! Until then, you can register to view our full ASHG program, including lightning talks, co-lab presentations and posters.

Learn more about highly accurate long-read sequencing and how it plays a role in human biomedical research.

Do you have questions about HiFi sequencing and how it can advance your research? Connect with a PacBio Scientist.

Read More »

Friday, October 29, 2021

Our Latest SMRT Grant Winner: HiFi Sequencing to Understand Microbial World


2021 Microbial SMRT Grant image
At PacBio, we understand the profound importance of microbial research. As the foundation of the biosphere and major determinants of human health, microbes claim a primary, fundamental role in life on earth. Microbes account for most of the diversity of life on our planet, yet fewer than 1% have been identified. To understand this ever-evolving world, scientists need the unbiased, accurate, and comprehensive information that only PacBio long-read sequencing can provide.

Our commitment to research has been shown through our 2021 Microbial Genomics SMRT Grant Program. Applicants were invited to apply for a chance to win free sequencing by our Single Molecule, Real-Time (SMRT) technology that provides the highly accurate long reads – HiFi reads – necessary to capture any microbe in high resolution.

We’re proud to announce the winner of this year’s 2021 Microbial Genomics SMRT Grant Program. While there were hundreds of submissions this year from all around the globe, only a select few could be chosen. Congratulations to these innovative scientists leading the future of microbial research.


Winner: Mark Nicol

Mark Nicol
Institution: The University of Western Australia, Perth, Western Australia, Australia

Project Goal: Species-level profiling of the upper respiratory microbiota to predict asthma in young children

We have previously done several studies using short-read 16S sequencing to characterise respiratory tract microbial communities in children with pneumonia and chronic lung disease. A major frustration has been the inability to obtain species-level information on taxa associated with illness. Species level discrimination is key in this niche, pathogenicity resides at the species, not the genus level. We are optimistic that the accurate species-level profiling which we will obtain from PacBio HiFi data will give us novel insights into the pathogenesis of wheezing in young children, and help us identify microbial biomarkers of risk of progression to asthma

— Mark Nicol

The respiratory microbiome in early life has been linked to the later development of asthma. However, associations are at the genus level, with poor understanding of how individual bacterial species may predispose to wheezing. With the help of PacBio long-read sequencing, Mark and his team hope to determine species-specific associations with the wheezing phenotype.

Sequencing for this project will be provided by the PacBio Certified Service Provider Maryland Genomics.

Maryland Genomics logo


Runner Up: Stephen Techtmann

Stephen TechtmannInstitution: Michigan Technological University, Houghton, Michigan, USA

Project Goal: Division of labor in plastic processing microbial communities

We’re excited to be working with PacBio to generate high quality assemblies of the members of these microbial consortia. Having complete genomes from these metagenomes will help us to better characterize how these communities are breaking down the plastic inputs so that we can better engineer these communities to increase efficiency. These high quality assemblies will help us to find novel solutions to deal with plastic waste and inform how we might be able to use these microbial communities for food production.

— Stephen Techtmann

Plastic pollution poses major issues for our environment. HiFi sequencing is opening doors to potentially develop a series of enriched microbial consortia that are able to grow to high densities using thermal and chemically treated plastic waste and rapidly breakdown deconstructed plastics. Stephen (@stechtmann) and his team hope that high quality metagenome assembled genomes can assist in modeling the flow of carbon through a community.


Congratulations to our 2021 Microbial Genomics SMRT Grant Program winners! And thank you to our co-sponsor for teaming up with PacBio to make these SMRT Grants possible. Explore the 2021 SMRT Grant Programs to apply to have your project funded.

If you’re interested in reading about the work PacBio does in Microbiology, explore our microbiology page dedicated to sequencing the microbial world with confidence.


Read More »

Monday, October 25, 2021

The Series that Made Our Summer – From Microbes to Metagenomes


microbiology agar cells colony and cultureThe dog days of summer might be behind us, but our four-part Microbial Genomics Webinar Series is sticking around as a focal point of the summer. From the end of July to the beginning of September, scientists shared with us how they have used highly accurate PacBio long-read sequencing to make new discoveries about bacteria, viruses, and the complex systems they inhabit and influence.

Here’s a recap of what was shared:


Part 1: Optimizing for Information: What Richer Data and Better Assemblies Reveal About Metagenome Structure and Function

Derek M. Bickhart, Ph.D., Research Microbiologist/Bioinformatician at the United States Department of Agriculture, and Dan Portik, Ph.D. (@DPortik), Bioinformatics Scientist at PacBio, kicked off the series talking about top tools and tips for metagenome sequencing and analysis on the Sequel Systems.

In “Deep Sequencing of a Sheep Fecal Sample with HiFi Reads and Hi-C Data,” Bickhart demonstrated how deep sequencing that combines HiFi reads and Hi-C data can enormously increase recovery of high-quality MAGs and connect plasmids and viruses to host strains. The presented HiFi sequencing data produced 127 high quality MAGs including 44 closed circles from a sheep fecal sample, which increased to 428 complete MAGs with Hi-C data.

Then, Portik dared viewers to “try this at home” in his session on HiFi sequencing metagenomics examples and workflows. Full instructions and example data are available in PacBio’s metagenomics GitHub

Part 2: Resolving Viral Evolution and Quasispecies Diversity with HiFi Sequencing

The second webinar could have been ripped right from the headlines, focusing on understanding HiFi sequencing methods for resolving viral diversity in complex systems—specifically that of COVID-19.

Eli Boritz, M.D., Ph.D., Chief, Virus Persistence and Dynamics Section at the National Institute of Allergy and Infectious Diseases, examined how combining fully-phased minor variant data with other data types provided insights into viral evolution, immune escape, and drug resistance in “Using HiFi Sequencing to Resolve SARS-CoV-2 Evolution During Acute COVID Infection.” Based on his findings, Borlitz suggested that the difference between the slow genetic drift of earlier sequences and the accelerated new variants of concern was the changing fitness constraints upon the virus.

Meredith Ashby, Ph.D. (@AshbyMere), Associate Director of Segment Marketing at PacBio, took the long view in her presentation Beyond COVID-19: The Long and the Short of Why Virologists Use HiFi Sequencing,” explaining how HiFi sequencing could phase entire viral genes or genomes, revealing quasispecies diversity within patients.

Part 3: Identifying Key Players in Host-Microbiome Interactions with High Resolution 16S Sequencing

Gut feeling takes on a whole new meaning when it comes to the research of Candice M. Brown, Ph.D. and Elaine Leung, Ph.D. Both women are studying how metagenome functions that impact human health can be driven by specific species or strains within a community, specifically in the gut microbiome.

In “Strain-level Profiling of the Gut Microbiome Reveals a Unique Microbial Signature in Experimental Sepsis,” Brown (@BrownLabWVU), an Assistant Professor in the Department of Neuroscience in the School of Medicine at West Virginia University, explored how the brain-gut-microbiota axis is affected by serious systemic inflammation, such as sepsis. She showed how highly accurate long-read sequencing of extended 16S amplicons enables identification of metagenome community members at higher taxonomic resolution than short read methods.

Leung, an Associate Professor at Macau University of Science and Technology, studies how the gut microbiome impacts drug efficacy. “Altering the Gut Microbiota to Activate Anti-PD-1/ PD-L1 Immunotherapy,” looked at the Leung Lab’s research into whether gut microbiome modulation via a ginseng compound combined with αPD-1 mAb could affect immunotherapy drug responses in Non-Small Cell Lung Cancer patients.

Part 4: Revealing Mechanisms of Bacterial Virulence and Adaptation with PacBio SMRT Sequencing

From the farm to the infirmary, attendees of the final webinar in the series learned how hard-to-assemble domains and plasmids impact important biological traits, including pathogen virulence and anti-microbial resistance. With “Identifying Virulence Factors in Black Rot with Long Read Sequencing,” Zoe Dubrow, Ph.D. (@ZoeDubrow), Delivery Technology Scientist at Pairwise, started things off by sharing the importance of both high accuracy and long read length for generating closed bacterial assemblies, as shown by example of Xanthomonas, a genus of Proteobacteria that causes black rot in cabbage.

Lynn Bry, M.D., Ph.D. (@see_anemone), Director of the Massachusetts Host-Microbiome Center at Brigham & Women’s Hospital and an Associate Professor of Pathology at Harvard Medical School, then emphasized the unique advantages of highly accurate PacBio long-read sequencing for pathogen surveillance and tracking in hospital settings.

Summer 2021 has been a whirlwind of events, announcements, and planning for the future. In the world of microbes and microbial DNA, there is still a ton in store. Stay tuned for an announcement on our most recent Microbial SMRT Grant winner, keep up to date on ASHG and check in regularly for updates about how HiFi sequencing is fueling discovery.

To watch the series OnDemand, follow these links: Part 1, Part 2, Part 3, Part 4

Read More »

Wednesday, September 29, 2021

Long-read HiFi Sequencing is Helping Researchers Tackle Biggest ALS Challenges


neuroscience long-read genomic sequencingSeven years after the ALS Ice Bucket Challenge soaked the world, the pace of discovery in sporadic amyotrophic lateral sclerosis has increased tremendously, with more than $115 million dollars in donations funding research that has led to the identification of several genes implicated in both familial and sporadic cases of the neurodegenerative disease.

While the social campaigns have generated much needed awareness around the disease, there are other challenges – one of which can be addressed with long-read sequencing.

As detailed in a new, interactive case study, PacBio SMRT Sequencing is helping researchers at the University of Washington unravel repeat regions of key genes linked to ALS and other disorders.

“We’ve made great strides over the past two decades in identifying genes and loci involved in ALS, mainly via GWAS studies,” said Paul Valdmanis (@pvaldmanis), assistant professor of medical genetics.


“We’re now at an exciting time where we have these new technologies available to allow us to identify novel risk factors. Through single cell sequencing and long read sequencing, in particular, we can ID some of these regions that were previously hidden or intractable to short read sequencing.”


Starting the search

Tandem repeats (and variable number tandem repeats, VNTRs) are snippets of DNA that are repeated multiple times within a gene, anywhere from a handful of times to more than a hundred. Sometimes these repeat sequences expand to long stretches, and these expanded repeats have been implicated in many diseases, including 40 linked to neurological disease.

Some of the open questions about these repeats include:

● How do they expand from a short repeat copy with four or five CAGs to an allele of over 60 base pairs?

● Does it make a difference where the repeat occurs–the beginning, middle, or end of the gene?

● What role does the internal sequence of these repeats play in disease pathogenesis?

In their search for answers, Valdmanis and postdoctoral fellow Meredith Course started with a multigenerational family that had several cases of ALS. Many of the family members had variants of a gene (FUS) linked to the disease, but not all of them exhibited symptoms. Why? Is there an additional genetic modifier that influences pathogenesis?

Using old-school linkage study approaches, the Valdmanis Lab identified a region on Chromosome 18 that also seemed to play a role. All members of the family affected by ALS shared a 4 megabase segment of DNA within this region. So, they took a look inside this region to see if there were other risk factors. Then, they zoomed in further, using HiFi sequencing as their magnifying glass.

Chromosome 18 ALS research


Homing in on the culprit

Through HiFi sequencing, researchers pinpointed a 69-bp VNTR in the WDR7 gene that was found to be enriched in individuals with ALS. The reference genome has about 6 copies of this repeat, but each individual in the ALS family had more than 30 copies.

They also performed multiplexed barcoded sequencing to resolve the complete internal structure of the WDR7 repeat in 288 geographically diverse individuals, and found striking variability in both repeat length and internal nucleotide composition. Some of the 69 bp repeat motifs were specifically present or absent in certain geographic populations.

They created maps to help visualize the data, and started to notice patterns emerging.

“Every time we looked at this repeat, we learned more,” Valdmanis said.

They were able to identify features associated with repeat expansion dynamics, the mechanistic consequences of repeat expansions to ALS susceptibility, and the structure of repeats in geographically diverse populations.

Further investigation of 15 samples from the Human Genome Project that had undergone long read phased sequencing suggested that the WDR7 gene was not alone in terms of the extreme variability in the length of its tandem repeats. They explored many other genes, including NWD2, VPS53, SLC22A1 and ART, and discovered various categories of repeats.

“We truly believe that long-read sequencing of tandem repeats can provide a lot of information about both human evolutionary events as well as risk factors for neurodegenerative disease. And we believe that VNTR expansions can represent novel disease risk factors not only in ALS but in other neurodegenerative diseases as well.”

Read our newest groundbreaking case study to learn more: ‘How SMRT Sequencing Helped Researchers at University of Washington Uncover a Tandem Repeat Linked to ALS

Interested in learning more about our technology and ALS research?

Read our blog: Scientists Use PacBio Sequencing to Discover Likely Pathogenic Structural Variants Linked to ALS

Watch Marka van Blitterswijk from the Mayo Clinic present, ‘Applying Targeted Long-read Sequencing to Assess an Expanded Repeat in C9orf72

See Meredith Course from the University of Washington present, ‘The Evolution and Function of a Large Tandem Repeat Associated with ALS’.

Visit our Neuroscience Research page to learn how PacBio sequencing provides a comprehensive understanding of the genetic basis of neurological disease.

Read More »

Thursday, September 23, 2021

ESHG 2021: How HiFi Sequencing is Closing the Gaps in Rare Disease Research


It’s a challenge that has haunted rare disease researchers for years: how to increase solve rates in rare and Mendelian disease. Currently, the genetic cause of more than half of rare disease cases worldwide remain unexplained.

In a series of talks and posters presented at the 2021 annual meeting of The European Society of Human Genetics (#ESHG21), PacBio experts and users described how HiFi sequencing could help close the gap by providing more comprehensive, accurate and high-definition coverage of the gaps in the human genome.

Here is a summary of the discussions that took place and the posters that were presented:


Long-Read Genome Sequencing for the Molecular Understanding of Rare Disease and Neurodevelopmental Disorders



In the first workshop, Susan Hiatt (@suzieqhiatt) from HudsonAlpha Institute for Biotechnology discussed how she and her team used HiFi sequencing in their rare disease research to discover genomic variation missed by whole-exome or genome sequencing studies using short reads.

PacBio’s Chief Scientific Officer Jonas Korlach also provided a summary about how advancements in sequencing technology, coupled with improved analytical tools and databases, have made it possible to finally examine hard-to-reach regions of the human genome, with huge implications for rare disease research.


“PacBio continues this march of technology evolution, and soon more than half, and perhaps up to two-thirds of unexplained rare disease cases can be explained through HiFi sequencing,” – Jonas Korlach

He went on to say: “We believe that high quality whole genome sequencing is the future of medicine and that this will be the first of many impactful demonstrations.”


Learn more about the work here.


Targeting Clinically Significant Dark Regions of the Human Genome with High-accuracy, Long-read Sequencing

ESHG Poster Targeting Clinically Significant Dark Regions of the Human Genome

In a poster presentation, researchers at PacBio showed how there are many clinically important genes in “dark” regions of the human genome. They explained how these often exist because of a paucity of NGS coverage or mapping difficulties, often complicated by the presence of various repeat elements or segmental duplications.

View the poster to see how long-read sequencing coupled with a long-PCR targeted enrichment method has the potential to illuminate these dark regions, using examples from CYP21A2, responsible for congenital adrenal hyperplasia, and GBA, responsible for Gaucher’s disease.


Resolving Complex Pathogenic Alleles Using HiFi Long-range Amplicon Data and a New Clustering Algorithm


ESHG Poster 2021 Resolving complex pathogenic alleles using HiFi long-range amplicon data and a new clustering algorithm

In another poster presentation, experts outlined how many genetic diseases are mapped to structurally complex regions containing highly similar paralogous alleles (>99% identity) that span kilobases and how, as a result, comprehensive screening for pathogenic variants is incomplete and labor intensive using short-reads or optical mapping.

View this poster to find out how long-range amplification and PacBio HiFi sequencing can fully resolve and phase a wide range of pathogenic variants with the help of a new amplicon analysis tool, pbAA.


Full-Length Sequencing of CYP2D6 Locus with HiFi Reads Increasing Genotypes Accuracy


ESHG Poster 2021 Full-length sequencing of CYP2D6 locus with HiFi reads increasing genotypes accuracy

And, in the final poster presentation, there was an insightful deep dive into personalized medicine. This poster reviews new discoveries around how the highly polymorphic CYP2D6 gene impacts the metabolism of 25% of the most prescribed drugs on the market. Experts reviewed this poster to inspire a discussion around how accurate identification of variant CYP2D6 alleles in individuals is necessary for personalized medicine.

View the poster to see how HiFi sequencing coupled with long-PCR targeted enrichment has successfully characterized 22 samples from a pharmacogenomics reference panel.


Comprehensive Detection of Variants in Unsolved Rare Disease Studies with PacBio HiFi Reads


Lastly, experts discussed how PacBio HiFi reads (99.9% accuracy, 15-25 kb) enable comprehensive variant detection in human genomes, extending to repetitive regions of the genome not accessible with short-read WGS (srWGS). It was revealed that HiFi reads match or surpass srWGS for single nucleotide variant and small indel detection while also improving detection of structural variants (SVs), with recall far exceeding that of srWGS.

This talk showcased how HiFi can be applied in a large-scale, reproducible way using an automated workflow, and how it performed on 80 rare disease cases unexplained by srWGS.


All in all, ESHG 2021 was full of insight, discovery and hope. With HiFi sequencing, we are excited about what the future holds.

If you would like to watch the workshop recording on demand, go here.

Or, if you’d like to learn more about highly accurate long-read sequencing and how it plays a role in human biomedical research, visit us here.

Read More »

Wednesday, September 8, 2021

UW Scientists Resolve Key Segmental Duplication Region with PacBio Sequencing


Neanderthal monument


A new Nature Communications paper shows how scientists continue to make progress elucidating some of the most complex regions of the human genome by deploying long-read PacBio sequencing technology. In this case, lead author PingHsun Hsieh (@phhBenson), senior author Evan Eichler, and collaborators at the University of Washington resolved the TCAF gene locus and identified more than 100 kb that had been missing in the human reference genome.

Since the publication comes from the Eichler lab, it’s no surprise that the target genes in this project emerged in a segmental duplication (SD) region. The TCAF genes — which encode TRP channel-associated factors related to thermal sensing in a type of neuron — “originated from an ancient gene duplication event at the basal of mammalian phylogeny and remained single-copy genes throughout much of their evolution,” the scientists report. In humans, duplications of this region in the past 1.7 million years have led to more copies of TCAF1 and TCAF2.

Until now, this locus of the genome has remained intractable. “In the human reference genome GRCh38, TCAF1 and TCAF2 are embedded and span within a complex region of large, highly identical SDs (>99.5%) consisting of >250 thousand base pairs (kbp) in sequence and an annotated gap at chromosome 7q35,” the authors note.


Filling A Pesky Gap

Copy number variation of TCAF SDs

Copy number variation of TCAF SDs in a collection of diverse human and nonhuman samples. From Hsieh, P., Dang, V., Vollger, M.R. et al. Nat Commun 12, 5118 (2021)


In this project, the team paired PacBio sequencing with large-insert bacterial artificial chromosome clones to resolve the entire locus in eight humans as well as in chimpanzee, gorilla, and rhesus macaque, generating 15 haplotypes of the region and even comparing their results to those seen in ancient human genomes. They also used the Iso-Seq method to analyze gene expression in seven different tissues.

“We systematically explore the haplotype structure of the TCAF locus in order to study its diversity, annotate the genes, and infer its evolutionary history in the context of selection,” the scientists report.


“This study is one of the detailed genetic investigations of human-specific SDs shedding potential new insights into structural adaptations important in thermal regulation.”


Sequencing results revealed that TCAF paralogs were more than 99.7% identical, with sizes ranging from 10 kb to 60 kb. They also filled that pesky gap in the human reference genome by identifying the missing 103,616 bp. The team focused on haplotypes of the region. While the non-human primates had just one copy each of the TCAF1 and TCAF2 genes, the 12 resolved human haplotypes were quite different. “We identify five distinct haplogroups that carry one to three copies for the SD cassette, which range from 145–406 kbp in length,” they write.


Isoform Diversity Sheds Light on Ancestral Diversity

These haplotypes allowed the team to dive into annotation and analyze isoform diversity. Using the Iso-Seq method, they produced more than 480,000 full-length, non-chimeric transcripts from analyses of six human tissues and more than 50,000 from a chimpanzee cell line. In humans they found considerably more isoform diversity for TCAF2 than for TCAF1.

Perhaps most strikingly, though, the scientists found evidence of contrary patterns of selection.

“Our data support a model of two distinct forces of natural selection possibly operating on the same locus over the last half million years of hominin evolution,” they report.

“We propose that diversifying or balancing selection is likely acting in at least some human populations, particularly out-of-African populations such as Native Americans, to maintain and expand haplotype and structural diversity.”

The ancient human samples told a different story.

“In contrast, Neanderthal and Denisovan show a paucity of genetic variation, and while the sample size is still limited, this observation is unlikely to change with the sequencing of additional archaic genomes,” the scientists add. “We hypothesize that positive selection has reduced genetic diversity at the TCAF locus in these archaic hominin lineages.”

Interested in learning more about Iso-Seq Analysis? Go here.

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Monday, August 23, 2021

SMRT Grant Winners: When Accuracy Matters Scientists Choose HiFi Sequencing


At PacBio, we are passionate about accuracy in sequencing data. Our commitment to ensuring reliable results is why our HiFi reads are better than 99.9% accurate. Combined with the length of those reads — up to 25 kb — and it’s no wonder that our sequencing data generates complete, contiguous, and correct assemblies for even the most complex genomes.


While we’re proud of these technical accomplishments, our favorite thing is seeing how HiFi reads empower scientists to make new discoveries and reach novel insights. To that end, we launched our HiFi for Accuracy SMRT Grant program earlier this year and now we’re pleased to announce the winners.

From a pool of hundreds of submissions, we selected three outstanding winners from around the world who will use HiFi sequencing provided by local service providers to advance their fields of study. Congratulations to these intrepid scientists and recipients of our HiFi for Accuracy SMRT Grant:


HiFi Sequencing to Understand Rare Diseases

Claudia Gonzaga-Jauregui of the National Autonomous University of Mexico

Winner: Claudia Gonzaga-Jauregui (@cgonzagaj)

Institution: International Laboratory for Human Genome Research, National Autonomous University of Mexico

Project goal: Understand genetic disorders that remain unanswered even after deep characterization with other molecular tools and sequencing platforms.

Many people’s rare genetic diseases remain unsolved even after applying molecular technologies like chromosomal microarray and exome sequencing. Implementing innovative technologies like PacBio long-read sequencing to look at variation genome-wide in rare disease research offers the opportunity to look at variants beyond the constraints of other technologies and help shed light on these medical mysteries. I am grateful to PacBio for awarding my new laboratory a SMRT Grant to use their technology to help enable rare disease diagnostics in Mexico.”

– Claudia Gonzaga-Jauregui


Sequencing for this project will be provided by the PacBio Certified Service Provider DNA Sequencing Center at Brigham Young University.



HiFi Sequencing to Understand the Evolution of an Iconic Species

Sven Winter of Senckenberg Biodiversity and Climate Research Centre

Winner: Sven Winter (@zoologysven)

Institution: Senckenberg Biodiversity and Climate Research Centre

Project goal: Generate high-quality assemblies of two giraffe species to facilitate analysis of structural differences

“PacBio HiFi sequencing will facilitate the detection of structural variance among giraffe genomes with high accuracy.”

– Sven Winter

A Giraffe in Samburu National Park, Kenya. © Julian Fennessy


Sequencing for this project will be provided by the PacBio Certified Service Provider CCGA.




HiFi Sequencing to Explore how Metagenomes Impact Infectious Disease

Charlene Kahler of the University of Western Australia

Winner: Charlene Kahler (@charlene_kahler)

Institution: University of Western Australia

Project goal: Conduct metagenomic analysis of oropharyngeal samples collected from individuals carrying meningococcal disease to identify factors involved in infection

“HiFi sequencing is the perfect technology for undertaking 16S microbiome survey to find signatures in the oropharyngeal microbiome that aid or prevent meningococcal colonization.”

– Charlene Kahler


Sequencing for this project will be provided by the PacBio Service Provider BIOTOOLS




Congratulations to our HiFi for Accuracy SMRT Grant winners! And thank you to our co-sponsors for teaming up with PacBio to make these SMRT Grants possible. Explore the 2021 SMRT Grant Programs for future opportunities to have your project funded.



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Friday, August 6, 2021

Young Investigators Share Stellar Science, Career Advice and Bioinformatics Tools at SMRT Leiden 2021


SMRT Leiden Virtual Event 2021

They spoke about omentum, chemosynthesis, chromothripsis, and… Tasmanian devils? This year’s virtual two-day SMRT Leiden Scientific Symposium and Informatics Developers Meeting was certainly educational.

With the pandemic and increased difficulty in being able to connect in person, we wanted to provide a forum for young investigators, post docs, and faculty to come together and share their research experiences during these abnormal times. The result? 27 speakers—the majority of whom were young investigators—sharing data and discoveries, and their advice for early-career scientists.

There was a great spectrum of presentations. The first keynote featured fun facts about Dominette (first Bos taurus to have her genome sequenced) and efforts to create a cow pangenome. Hubert Pausch of ETH Zürich discussed the downfalls of reference-guided variant discovery and the benefits of genome graphs to overcome some of the biases of linear mapping.

The second keynote by 2019 Human Genetics SMRT Grant winner Tychele Turner (@tycheleturner) highlighted the value of HiFi reads in investigating neurodevelopmental disorders, including 9p minus disorder and autism. Turner said HiFi reads should become the new paradigm because of their ability to detect more variants, reveal novel variation, and phase even the most complex genes.


“PacBio long-read sequencing is ushering in a new era in human genomics.” — Tychele Turner, PhD., Washington University Genetics

In other sessions, early data from another SMRT Grant winning project about neurological diseases with complex structural rearrangements was reviewed. Matthew Hestand of Cincinnati Children’s Hospital Medical Center was joined by University of Louisville researcher Corey Watson (@ctwatson29), who spoke about characterizing immunoglobulin haplotype diversity and its influence on the antibody repertoire, and Gloria Sheynkman (@GSheynkman) of the University of Virginia, discussed the integration of long-read RNA-Seq and mass spectrometry.

In another session, Harald Gruber-Vodicka (@GruberVodicka) of the Max Planck Institute for Marine Microbiology imparted some useful advice about sample preparation and assembly of the tiniest samples, and Jannat Ijaz (@sciencejannat) of the Wellcome Sanger Institute described catastrophic genome fragmenting events that can occur in cancer, and how she pieced together 900 fragments from esophageal organoids.

Lastly, in view of the pandemic and the challenges associated with it, we invited Melissa Smith (@SmithLab_UofL) to speak about work in her new lab at the University of Louisville, covering both COVID-19 surveillance and research into one of the biggest hurdles in HIV therapy, HIV “reservoirs.”


Pursue Your Passions


SMRT Leiden 2021 Speakers


In addition to getting a look at the research being done around the world, we understand that the pandemic has been an unprecedented time for early-stage researchers. In view of these challenges, we decided to host a session dedicated to career advice, guidance, and open discussion. This ended up being one of the event’s most popular sessions, featuring valuable learnings from speakers.

Here are a few examples of the advice they shared:

On the art and craft of being a scientist, finding a mentor, and how to lead:

Choose your mentor over your scientific subject. Learning the craft and being the best scientist you can be is most important, and can then be applied to the subject you love. Also, don’t stymy your creativity. Practice it. Try to have one new idea every day—it doesn’t even matter if it involves science. — Jonas Korlach, PacBio CSO

Think about what kind of leader you want to be. You learn how to be a good scientist or bioinformatician, but no one tells you how to lead a group. Consider taking a course, or learn from your favorite leaders. — Susan Kloet, Leiden University Medical Center

On recognizing the talents we each possess, and using those talents to succeed in science:

Drive yourself. Don’t compare yourself to anyone else. Don’t rely on anyone else to set expectations and hold yourself accountable. Remove the word ‘should’ from your career vocabulary. — Melissa Smith

Be open to trying something new. And don’t be cowed by others’ successes. They’ve encountered challenges too. — Tychele Turner, PhD., Washington University Genetics

On being open to revision and excited by the chance to iterate:

Be happy if you see your papers returned covered in red (edits) – it means your supervisor cares. Don’t be frustrated. Take their advice and use it to improve. — Hubert Pausch, Prof. Dr., ETH Zurich

And, last but not least – on passion:

You can still make great contributions to science outside of academia. Even extracurricular activities like roller derby can teach you key soft skills like leadership and organization. Pursue all your passions. — Sarah Kingan, Senior Product Manager at PacBio

And, don’t forget to leave the lab every once in a while.


Bioinformatics: Top 10 Tools

SMRT Leiden 2021 complete human genome

Another exciting part of the event concentrated on bioinformatics. Serendipitously, these bioinformatics sessions coincided with the Telomere-to-Telomere Consortium’s release of the first ‘complete’ human genome. In her keynote address at SMRT Leiden 2021, consortium member Arang Rhie (@ArangRhie) gave a behind-the-scenes look at how the team sequenced a human genome in its entirety for the first time ever in history. This included characterizing the final unresolved 8% of the genome.

The other keynote by Tobias Marschall (@tobiasmarschal) of Heinrich Heine University Düsseldorf provided an interesting review of haplotype-resolved assemblies and the Human Genome Structural Variation Consortium.

Still more, speakers presented the tools they’ve developed to optimize HiFi reads, including:

Merfin – k-mer-based assembly and variant calling evaluation for improved consensus accuracy (Arang Rhie)
PanGenie – algorithm that leverages a pangenome reference built from haplotype-resolved genome assemblies in conjunction with k-mer count information from raw, short-read sequencing data to genotype a wide spectrum of genetic variation (Tobias Marschall)
SQANTI3 – an automated pipeline for the classification of long-read transcripts that can assess the quality of data and the preprocessing pipeline (Rocío Amorín de Hegedüs @rocioadh)
tama (Transcriptome Annotation by Modular Algorithms) – software designed for processing Iso-Seq data and other long-read transcriptome data (Richard Kuo @GenomeRIK)
pbaa (PacBio Amplicon Analysis) – separates complex mixtures of amplicon targets from genomic samples to cluster and generate high-quality consensus sequences from HiFi reads (Zev Kronenberg @zevkronenberg)
bellerophon – analyzes MHC typing and other low-complexity gene amplicon data; performs allele calling while detecting polymorphic sites within the sequences and removing potential chimeric sequence variants (Yuanyuan Cheng @Yuanyuan929)
svpack – tools for filtering, comparing, and annotating structural variant (SV) calls in VCF format (Aaron Wenger)
JumboDB – tool for de Bruijn graph construction (Anton Bankevich @AntonBankevich)
uLTRA – tool for splice alignment of long transcriptomic reads to a genome, guided by a database of exon annotations. (Kristoffer Sahlin @krsahlin)
LeafGo – workflow to rapidly produce high-quality de novo plant genomes (Luca Ermini @ermini_luca)

By and large, SMRT Leiden 2021 was packed full of valuable discussion, behind-the-scenes info, and exciting revelations about the research being done via HiFi sequencing. Each session is available to watch on demand. Register for free now, before it’s too late.

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Monday, August 2, 2021

Data Release: Human Microbiome Samples Demonstrate Advances in HiFi-Enabled Metagenomic Sequencing


As technology developers, one of our greatest joys is seeing how customers take our sequencing tools and deploy them for innovative and compelling new projects. Metagenomics has been one of those areas: our customers have recently been demonstrating the significant performance improvements enabled by our HiFi metagenome sequencing data and analysis pipelines.

But since much of that work is protected by HIPAA regulations or has not yet been published, we are now releasing a metagenomic data set to help scientists see how HiFi data can make a difference for these types of studies. This information is now available for review and analysis and can be used with existing tools or to help develop new ones.


Human fecal microbiome samples The data set was generated from four fully consented, pooled human fecal microbiome samples made available through The BioCollective. Two samples came from vegan donors and two from omnivore donors, allowing us to see how diet influences gut microbiota. The pooling process, which creates a reference material by pooling samples from multiple donors (in this case four adults), leads to a more complex sample and a richer data set than can be obtained through mock community approaches. It also gives a more consistent composition than samples from an individual donation.


Long-Read Sequencing Produces Rich Profiling Information

BioCollective functional annotations HiFi sequencing gave us nearly 2 million reads per sample, with mean read length close to 10 kb for each. Median quality for the sequencing data was Q39 for two samples and Q40 for two samples. We found that species composition was consistent within diets and different between diets. Of the 76 bacterial species detected, 14 were exclusive to the omnivore samples and 21 were only found in the vegan samples.

There are a lot of exciting things to unpack in this data set. First, it demonstrates that our data analysis pipelines produce rich functional profiling information. Unlike analyses of short-read data, about 90% of HiFi reads have at least one functional annotation, with reads typically having two to five annotations. For each sample run on a single SMRT Cell 8M, we generated more than 8 million total annotations.


BioCollective nucleotide identity
In addition, the data set highlights the advantage of high accuracy when assembling long-read data from metagenomes. These samples often contain closely related strains. A common cutoff for defining a distinct species is just 3%; if the difference between strains is less than the error rate, then the error correction process can erase the real differences needed to resolve and distinguish those strains.

BioCollective MAGs graphThis heightened ability to resolve strains is what drives the large number of high-quality metagenome-assembled genomes (MAGs) that can be recovered from a relatively small amount of HiFi data. For each sample, our assembly evaluation pipeline identified at least 56 — and as many as 69 — MAGs. The unique combination of high accuracy and long reads means that high-quality MAGs can be generated with less than 20-fold coverage, and many of those MAGs are represented in a single contig.

Listen to Daniel Portik talk about this new dataset in the first episode of our Metagenomics Webinar Series on demand here. We hope you get the chance to download the data and experience it for yourself.

Want to talk to us about this data set or have project ideas where you think HiFi data can make a difference? Hit us up on Twitter or reach out directly to our metagenomic specialists Meredith Ashby or Daniel Portik.

If you are interested in additional Metagenomics Webinars, register for upcoming episodes to learn about:
How to resolve viral evolution and quasispecies diversity mechanisms of bacterial virulence and adaptation,
Identifying key players in host-microbiome interactions with high resolution 16S sequencing, and
Revealing mechanisms of bacterial virulence and adaptation

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Friday, July 30, 2021

Announcing the Winners of Our Clinical Research SMRT Grant – Two Scientists at the Forefront of Discovery

Here at PacBio, we have had the privilege of awarding many SMRT Grants to intrepid scientists who believe that HiFi sequencing data can help them achieve their goals. Recently, we invited people to apply for our Clinical Research SMRT Grant for projects with a link to potential clinical utility. We believe these projects could benefit tremendously from the value of HiFi reads, which offer both high accuracy and long reads to reveal genomic insights often missed by short-read sequencing.

Narrowing these applications down to just one winner is always challenging, but this time we found it to be impossible. So, for the first time ever, we planned to give one award and wound up making two awards instead. We are thrilled to announce the winners— one scientist at the start of her career and one well established in hers. We couldn’t be prouder to support the work of these two outstanding women and the questions they seek to answer.

Please join us in congratulating Danielle Brandes and Jenny Taylor on becoming our latest SMRT Grant winners! Here’s a look at what they plan to do with their awards.


Danielle Brandes, PhD Student

Institution: Pediatric Oncology, Medical Faculty, Heinrich Heine University Düsseldorf

Project Goal: Discover structural variants related to pediatric acute lymphoblastic leukemia that have been missed by other technologies.

Danielle’s proposal piqued our interest for many reasons. Acute lymphoblastic leukemia (ALL) is the most common childhood cancer to-date. Scientists understand that cancer predisposition genes (CPGs) are part of the puzzle when it comes to genetic predisposition in leukemic patients. However, CPGs are just a piece of the story. A large portion of the genome is affected by structural variants. Unfortunately, when it comes to leukemia, little is known about how structural variations play a part.

Despite technical and analytical progress in the field of NGS, the landscape of structural variations remains largely unresolved. In this context, we are excited to see how PacBio HiFi long-read sequencing will complement our whole-genome optical mapping data set to elucidate potentially pathogenic SVs in our studies of acute lymphoblastic leukemia. This approach will give new insights on mechanisms of leukemic predisposition as well as to the spectrum of somatic structural variation in leukemia.

— Danielle Brandes

Danielle Brandes works in the lab as a PhD Student at Heinrich Heine University Düsseldorf.

Danielle’s team has performed whole-genome optical mapping (WGOM) to identify SVs in pediatric patient studies diseased with a high hyperdiploid or ETV6-RUNX1 translocated ALL. But, there is more to be done. Through HiFi sequencing, Danielle hopes to detect additional SVs that might have been missed by, or could complement, the WGOM data she has been gathering.

With the help of the SMRT Grant, we are excited to see how Danielle will be able to use HiFi sequencing to generate an individual comprehensive germline/leukemia genome in the pursuit of pathogenic SVs in CPGs and somatically acquired events in ALL studies.


Jenny Taylor, Associate Professor

Institution: University of Oxford

Project Goal: Use HiFi sequencing to resolve structural variants and phase variants for a few participants in the UK’s 100,000 Genomes Project as a demonstration of how this approach could potentially help address unsolved disease cases.

Our second winner, Jenny Taylor, is a seasoned scientist with years of experience in her field. Now, she is hoping to use PacBio’s technology to add value to specific samples collected as part of the 100,000 Genomes Project to further understand rare diseases.

Jenny Taylor (far right) with her team from the Biomedical Research Centre, University of Oxford.

“I am delighted to be awarded this grant from PacBio that may help our lab support Genomics England to increase understanding of undiagnosed diseases for some of those who have been referred to the 100,000 Genomes Project.”

— Jenny Taylor

The UK’s 100,000 Genomes Project completed whole genome sequencing for 73,880 genomes from rare disease patients. Jenny is hopeful that further research can be done to investigate the pathogenesis of some of the unsolved rare disease research cases to which they have access. With HiFi sequencing, she hopes to undertake comprehensive variant detection in 3 genomes to provide proof-of-principle for the PacBio platform.


Congratulations to these two outstanding scientists! We couldn’t be more excited to see what comes of these projects and are honored to sponsor each of these scientists in their pursuit of discovery.

And thank you to our co-sponsor, Icahn Institute for Data Science and Genomic Technology, for teaming up with PacBio to make this SMRT Grant possible. Explore upcoming SMRT Grant Programs to apply to have your project funded.


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Thursday, July 22, 2021

For Metagenomic Studies, HiFi Reads Deliver Higher-Quality Data


A new paper from scientists at the Max Planck Institute offers a great look at how HiFi sequencing delivers significantly improved results for metagenome studies compared to short-read data. In this project, HiFi reads led to higher-quality assemblies with less coverage and gave more insight into these complex microbial communities.

Bathymetric map of sampling locations

Bathymetric map of sampling locations

In the PeerJ publication, lead author Taylor Priest (@taylorpriest2), senior author Rudolf Amann, and collaborators report the analysis of 11 seawater samples collected from the Fram Strait, which connects the Arctic and Atlantic oceans and offers a unique view of how climate change is affecting marine ecosystems.


Long-Read Sequencing in Marine Ecosystems Impacted By Climate Change

They performed metagenome sequencing of all samples with short-read technology and analyzed three of them with HiFi reads using the ultra-low library protocol on the Sequel II System. For the PacBio sequencing, all three samples were pooled and sequenced together on a single SMRT Cell, leading to 4-6 Gb of HiFi data per sample.

The PacBio data set yielded 128 metagenome assembled genomes (MAGs) from about 15 Gb of total data collected. In contrast, the short-read data set was about 10 times that size, but produced just 218 MAGs, or fewer than twice as many.


“Of the species-representative MAGs recovered, those generated from the PacBio metagenomes had, on average, larger genome sizes, higher N50 values, and were less fragmented compared to those retrieved from Illumina metagenomes,” Priest et al. report.


The quality of the assemblies also allowed the researchers to simplify their metagenome assembly pipeline. Importantly, the authors note, “taxonomic reassembly was not performed for the PacBio dataset due to the high quality of generated MAGs from single metagenome assemblies.”

HiFi assemblies also showed strength for community composition analysis. In metagenomics, researchers often pull out the 16S rRNA gene sequences to identify the microbial members of a community. Unfortunately, short-read data is not well suited to this task.

“In this study, 84% of MAGs retrieved from the PacBio metagenomes contained at least one complete 16S rRNA gene sequence, highlighting another key advantage of using long Hifi reads.”

This advantage also extended to unassembled data. “A major restriction with short-read metagenomic sequencing is the limited capacity to accurately reassemble full length 16S rRNA genes,” the team notes. “With the advent of highly accurate long read sequences generated from PacBio sequel II (>99% accuracy), full length 16S rRNA genes can be retrieved from single reads without a need for assembly, thus circumventing previous limitations.”


HiFi Reads for Marine Metagenomes Reveals Phylogenetic Diversity


An analysis of these results gave the scientists an interesting look at microbial populations in an area where warming sea water is already having an impact. The recovered diversity encompassed 9 phyla, 11 classes, 27 orders, ∼51 families and ∼54 genera. The most species-rich taxa were the Flavobacteriales (41 species), Pseudomonadales (18 species) and Rhodobacterales (17 species).


Phylogenic diversity MAGs from Fram Strat

Phylogenetic diversity of metagenome assembled genomes (MAGs) from the Fram Strait.


This paper describes the team’s first use of HiFi sequencing for marine metagenomes, but it likely won’t be the last.

“We can conclude that HiFi read metagenomes derived from the PacBio Sequel II platform can greatly improve the number and quality of MAGs recovered, which will allow for further advancement in our understanding of the ecology of marine microbial communities,” they report.

Want to learn more about how HiFi Reads allow researchers to see metagenomes in high resolution? Visit our Microbial Applications page.

Have you heard about our Metagenomics Webinar Series?

Register now for the first episode in our series, highlighting what richer data and better assemblies reveal about metagenome structure and function. Or, stay tuned for the additional webinars in the series showcasing:
How to resolve viral evolution and quasispecies diversity mechanisms of bacterial virulence and adaptation,
Identifying key players in host-microbiome interactions with high resolution 16S sequencing, and
Revealing mechanisms of bacterial virulence and adaptation

Read More »

Wednesday, July 14, 2021

Iso-Seq Analysis Provides Insights into Feats of Physiology of Hibernating Bears


Hibernating bears have heart rates of 10-15 beats per minute, yet they do not develop congestive heart failure. Despite accumulating enormous amounts of fat and acquiring insulin resistance, they do not suffer metabolic diseases. And they maintain muscle strength in the near absence of weight-bearing activity.

Iso-Seq analysis provides insight into physiology of hibernating bears

Iso-Seq study has revealed differential tissue-specific isoform changes in brown bears

If we could crack these feats of physiology, perhaps we could apply the knowledge towards therapeutic targets for the prevention and treatment of numerous human diseases.

The Project that Shed Light on the Metabolic Mystery of Brown Bears

Washington State University researchers have come several steps closer to characterizing the hibernation phenotype by analyzing differential gene expression and tissue-specific isoform changes between active, hyperphagic, and mid-hibernation physiological states in the brown bear (Ursus arctos).

Led by the lab of Joanna L. Kelley (@joannalkelley), the team first identified more than 10,000 genes differentially regulated in adipose (fat), liver and muscle tissues between active and hibernating states

In a new preprint, they report the extent of tissue-specific isoform changes that take place during hibernation, revealed via PacBio long-read isoform sequencing (Iso-Seq).

The project, which was supported by a PacBio SMRT Grant, involved sequencing and analysis of tissues from three bears using the Iso-Seq method for full length RNA transcripts. The Sequencing & Genotyping Center at the University of Delaware sponsored the grant and processed the RNA with the help of the center’s director, Bruce Kingham (@bkingham).


“Single Molecule, Real-Time Sequencing Iso-Seq is ideal for identifying the full-length isoforms that are differentially expressed between seasons,” the authors wrote.


By combining the Iso-Seq data across samples and replicates, they obtained a total of 6.1 million full-length HiFi reads. After running the long reads through analysis, mapping to the reference genome, and filtering for library artifacts, they obtained 76,071 unique, full-length isoforms ranging from 150 bp – 16.5 kilobases (kb).

Isoform classification distribution by tissue

They merged these isoforms with the existing reference transcriptome (which contained 30,263 genes encompassing 58,335 transcripts) and found a total of 31,829 genes encompassing 107,649 transcripts, thus greatly increasing the number of known transcripts.


“Importantly, this merging of the reference transcriptome with the full-length transcriptome originating from samples of interest improves the reference and could lead to the discovery of differential isoform usage (DIU) that would otherwise be missed,” the authors note.


Tying Hibernation Biology to Human Health

Analysis of the data showed that metabolically active tissues vary dramatically in their isoform usage and underscored the complexity and importance of adipose as a dynamic tissue during hibernation. It demonstrated that both transcription and RNA processing play concerted roles.

“While differentially expressed genes have shed light on hibernation biology, determining genes where functionally distinct isoforms change between seasons is the next essential biology to uncover,” the authors wrote.
Tying hybernation biology to human health

“Our study provides an unprecedented view into hibernation biology through the lens of RNA processing by producing a dataset that improved the annotation of the brown bear genome and reinforced the important role adipose plays in hibernation.”

Researchers have suspected that aspects of hibernation physiology might be applicable to solving certain types of human disease. Until now, however, little has been known about the role of differential isoform expression between a bear’s hibernation and active states.

SMRT Sequencing has allowed researchers at WSU to generate the most comprehensive analysis of isoform usage. This opens doors for further research into how things like seasonal insulin resistance and sensitivity, obesity, and urine production in bears during hibernation and activity can help inform targets for disease solutions in humans.

See what happened when PacBio scientist (and now bear enthusiast) Michelle Vierra (@the_mvierra) joined the WSU team as they collected samples for the project from bears at the WSU Bear Center. She wrote an account of the experience on Medium, and filmed her meeting with Willow the bear in the video below.


For more information about the SMRT Grant and how you can apply – go here.

If you’re interested in how to identify novel gene isoforms or have questions about Iso-Seq analysis, visit our RNA Sequencing page.

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Tuesday, July 13, 2021

Iso-Seq Analysis Enthusiasts Share Research Wins at Virtual Social Event

Been itching to talk about your latest single-cell experiments, your favorite differentially expressed isoforms, or your latest and greatest software for visualizing alternative splicing, but thwarted by a worldwide pandemic preventing in-person scientific events? Iso-Seq Social Club Virtual Event

We were too, so we organized a virtual social club to easily enable scientists to geek out together. And we weren’t disappointed by our first event, which attracted dozens of self-proclaimed Iso-Seq analysis geeks and other curious researchers to share their work (published, unpublished and in progress) and discuss the benefits and challenges of incorporating long-read transcript sequencing into their research.

Welcome to the Iso-Seq Analysis Universe

PacBio’s own Iso-Seq analysis expert, Elizabeth Tseng (@magdoll) kicked off the Iso-Seq Social Club with an introduction to the method, which uses PacBio’s HiFi reads to characterize full-length transcript isoforms. The Iso-Seq method has been used to identify aberrant splicing in genetic diseases, characterize alternative promoter usage in cancer, and is making its way into the single-cell space for studying subregions in postnatal mouse brains and even ant brains!

But none of these studies are possible without proper tools, and as attendees learned, bioinformatics tools made specifically for long-read transcriptome data is a bustling field.

Francisco Pardo-Palacios (@FJPardoPalacios) and Ángeles Arzalluz Luque (@aarzalluz_), both from the Ana Conesa lab at Universitat Politècnica de València, presented the trilogy of SQANTI, IsoAnnot, and tappAS, which takes the output from the PacBio Iso-Seq analysis through classification, functional annotation, and differential analysis. Many of these tools are now becoming the standard workflow for Iso-Seq studies.

TES-switching gene Myef2 report

Swan is a Python library that can visualize complex isoforms, identify differential isoform usage and splicing patterns. It can work on both bulk and single-cell data.

Fairlie Reese (@FairlieReese), a PhD candidate from UC Irvine, presented her tool, Swan. It provides a graphical representation of alternative splicing events, but can also be used to detect differential isoform usage and isoform switching events.

Iso-Seq data to study brown bears between hibernation and active state.

Using Iso-Seq data to study brown bears between hibernation and active state. Iso-Seq data revealed genes where the overall gene-level expression did not change, but there were differential isoform usage (DIU) and major isoform switching events.

The Hunt For Differentially Expressed Isoforms In Bears… and Brains

Using Iso-Seq data on brown bears during hibernation and active seasons, Joanna Kelley (@joannalkelley) associate professor at Washington State University, was able to discover that fat tissue had higher levels of differential isoform usage (DIU) compared to liver and muscle tissues.

“Genes that show no change in expression levels but show major isoform switching and differential isoform usage are the ones we’re most interested in, because those are isoforms that we can’t quantify in any other way,” Kelley said.

Jack Humphrey (@JackHumphrey_), a postdoc in the Towfique Raj lab at Mount Sinai, is using Iso-Seq analysis to study complex splicing in genes associated with Alzheimer’s disease risk. Humphrey shared data from 30 post-mortem isolated microglia they collected. He also presented the processing pipelines for annotating and classifying the Iso-Seq transcripts, with an emphasis on filtering potential library artifacts – an often neglected but critical aspect of any bioinformatics work. Using a combination of existing tools and custom filtering, Humphrey showed that the curated transcriptome is high-quality and has already revealed interesting splicing events not observed with short-read data.

Single-Cell Iso-Seq Method for Precision Oncology and Hematopoietic Lineages

Arthur Dondi (@ArthurDondi), a PhD candidate from ETH Zurich, is using single-cell Iso-Seq (scIso-Seq) to study ovarian cancer. Specifically, by characterizing full-length isoforms in the omentum (fatty tissue covering the abdomen), there’s a potential for discovering neoepitopes and therapeutic targets.

Dondi and collaborators employed the HIT-scIso-Seq technique, which employs TSO artifact removal and concatenation for cDNA molecules coming out of the 10X single-cell platform, and increased the number of reads per SMRT Cell 8M by six-fold. They are planning to query this rich dataset for differential isoform expression, novel isoforms and fusion discovery.

Vladimir Souza from University of Zurich is working on calling variants from Iso-Seq data, showing that using DeepVariant or GATK with specific parameters achieved the highest precision-recall. The goal of his project is to eventually link the variations to changes in ORF predictions.

Anita Scoones (@AnitaScoonesPGR), a PhD candidate from the Earlham Institute, is studying lineage bias during hematopoietic stem cell differentiation. She wants to use single-cell Iso-Seq analysis on their plate-based single-cell libraries, similar to how her lab mate Laura Mincarelli had used long reads to look at isoform differences in aging mice.

Single-cell Iso-Seq reveals high isoform diversity in lineage-negative subpopulations

Figure 3. Using single-cell Iso-Seq reveals high isoform diversity in lineage-negative subpopulations that are enriched in novel isoforms (NIC, NNC) compared to total bone marrow.

Anne Deslattes Mays (@adeslat) and Marcel Schmidt of Georgetown University had previously used bulk Iso-Seq analysis to show that lineage-negative cells in bone marrow have higher isoform complexity than lineage-positive cells. They are now pushing the question into the single-cell space: is isoform diversity uniform at the single-cell in lineage-negative cells? Applying the scIso-Seq method, they found striking differences between the total and lineage-negative bone marrow subpopulations, where lineage-negative cells had an overwhelmingly high number of novel isoforms and were enriched in spliceosome-associated genes. This suggests that alternative splicing in lineage-negative cells is attributed to cell-fate decisions of each cell subpopulation.

What’s Next For Iso-Seq Analysis?

The event ended with a lively discussion in which attendees discussed the need for bioinformatics tools that can handle large amounts of Iso-Seq data and create reproducible workflows that others can easily adapt. They also addressed the one-size-fits-all approach of using a single reference annotation and said a re-think may be in order.

“Maybe references should be qualified by the tissues or cell types of interest,” suggested Ana Conesa (@anaconesa). “How do we use all these novel isoforms to annotate the transcriptome?”

Mays agreed that “the best reference is self.”

In neuroscience, scientists have a poor idea of what makes a cell type-specific isoform, Humphrey said. The challenge is agreeing on what a definitive reference for each cell type would be, he added.

“We’re not done at just references,” Schmidt suggested. “We need to assign a function to these isoforms, even if it’s a regulatory one.” And Conesa said a system level of analysis is necessary.

Overall, the enthusiasm around Iso-Seq analysis is consistent. The promise of a properly defined transcriptome summarized the conversation and paves the way for future discussion.

Want to learn more? Register to watch an on-demand recording of the event, or check out these resources:
PacBio Applications and Workflows
RNA Sequencing with Iso-Seq Analysis
Procedure & Checklist

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Thursday, July 1, 2021

In Case You Missed It — Sequel IIe System Users Share Their First Time Experiences

The new kid on the PacBio block — The Sequel IIe System — has been receiving high marks from universities and sequencing centers around the world.

What’s it like using the instrument, which was introduced in October 2020? Several users have spoken about their experiences in a series of recent online events.

Launching PacBio Sequencing Services in a New Lab

Sequel IIe Webinars

Bringing SMRT Sequencing to the BlueGrass State

Melissa L. Smith (@SmithLab_UofL), spoke about her experience transferring her lab from New York City to the “PacBio naive” Bluegrass State in the Unleashing the Power of HiFi webinar.

Smith admitted she faced some initial challenges in establishing her lab. Chief among them were compute capacity, data storage, ancillary equipment and staff expertise. Luckily, she was able to leverage existing campus resources to overcome many of those hurdles.

As for the computing needs, “The Sequel IIe changed everything,” she said.

Her favorite feature? On-instrument data processing, which has solved many of her compute capacity and data storage challenges. Plus, it has eliminated the need to queue or compete for compute resources with others across campus.

“The data coming off it is already collapsed, error corrected, and just 50-100 Gb, compared to ~1 TB from Sequel II,” she said.

The Sequel IIe System is not only supporting her research into immunology and infectious disease, it’s also part of a sequencing core lab, and one of PacBio’s newest Certified Service Providers. In addition to the standard sequencing pipelines, the lab will be doing assay development, SARS-CoV-2 sequencing with the new HiFiViral protocol, and other customized sequencing solutions.

Powering a Wide Range of Sequencing Applications

At the SciLifeLab in Uppsala, Sweden, the Sequel Systems are used for a whole spectrum of applications, from de novo genome assembly to BACs, YACs and filling gaps, Olga V. Pettersson told webinar attendees. Her team has been working with PacBio sequencing since 2013, initially with the PacBio RS II, and they recently upgraded to a Sequel IIe System.

In 2020, they sequenced more than 200 non-model eukaryotic genomes (around 700 individuals total), with many reaching the high quality standards of the Earth BioGenome Project.

Pettersson is also a fan of the Sequel IIe System’s advanced computing capabilities, saying it has led to a 20-fold reduction in data storage needs. HiFi reads have also helped shed light on hard-to-access “dark” regions of the human genome, she added.

Q&A with Genomics Core Facility Directors

Pettersson also appeared on a panel of expert users in SMRT Sequencing as a Service – How to Bring Long-Read Technology to Your Core Lab.

She shared tips for sample prep, instrument handling, and business planning, as well as some of the advantages of the Sequel IIe System.

“When the DNA is sufficient, we always prefer to go with PacBio because it’s so much easier, with bioinformatics off the shelf, reads of higher quality, and no need for additional polishing,” Pettersson said.

Other panelists, including Bruce Kingham of the University of Delaware Sequencing and Genotyping Center, said the Sequel System and its HiFi reads have become “the platinum standard for long read sequencing,” with extremely high demand among their users.

“There’s really no other data type like HiFi,” added Charlotte Harris, research lab supervisor at Corteva Agriscience. “Throughput has been a huge win for us. It’s allowed us to take on these much larger and more complex projects, and really benefit our profit margins.”

Want to learn more? Attend the on-demand webinar to hear from Melissa L. Smith and Olga Pettersson how the Sequel IIe System is making it easier than ever before to get started with HiFi reads or add capacity.

Want to discuss the benefits of HiFi sequencing and the Sequel IIe System for your research? Connect with a PacBio Scientist.

Interested in becoming a service provider? Visit the Sequencing for Service Provider page.

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Press Release

PacBio Grants Equity Incentive Award to New Employee

Friday, December 3, 2021