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A new program at ASHG this year came from the CoLaboratories, or educational theaters featuring 30-minute talks on a variety of clinical, laboratory, and data analysis topics. We were honored to present in the inaugural CoLabs today, with PacBio scientists offering tips on CRISPR/Cas9 enrichment as well as long-read whole genome sequencing for structural variant discovery.
Tyson Clark, director of applications development, gave a talk titled “Targeted Enrichment without Amplification and SMRT Sequencing of Repeat-Expansion Disease Causative Genomic Regions.”
He demonstrated a novel technique using the CRISPR/Cas9 system to target specific genes or elements in the human genome. Having an amplification-free method for this is particularly important for challenging targets, such as the many repeat expansions known to cause disease.
Combining this strategy with SMRT Sequencing allows for highly accurate, uniform, and complete sequencing of complex genomic regions that have proven intractable with short-read technologies. Clark reported successfully targeting and sequencing loci involved in several repeat expansion disorders — including HTT, FMR1, and ATXN10, among others — even when those regions had extreme GC content.
This work was also presented in an ASHG poster by Clark earlier in the week.
Staff scientist Aaron Wenger delivered a talk entitled “PacBio Long-Read WGS for Structural Variant Discovery.” As he noted, structural variant analysis has become increasingly important as the community realized that SNPs only explain a fraction of the DNA sequence differences between any two individuals, while structural variants represent the majority of those differences. These variants have been more difficult to detect, though, because of their size and complexity. Unlike short-read platforms, SMRT Sequencing can accurately characterize the vast majority of structural variants in a human genome, even at low coverage.
Wenger offered a look at updated structural variant calling protocols for the Sequel System, including how to tune parameters such as sequence coverage, and shared quick case studies.
Many thanks to all the ASHG attendees who joined the CoLabs to hear these talks!
The PacBio team hosted a luncheon workshop at ASHG yesterday titled “Population and Clinical Genetics Studies Using Long-read SMRT Sequencing.” Thanks to all the conference attendees who took time out of a very busy meeting to join us! If you couldn’t attend, we summarized the highlights below and will share recordings of the presentations soon.
Long-read Sequencing – for Detecting Clinically Relevant Structural Variation
Han Brunner, Head of Clinical Genetics at Radboud University Medical Center, kicked off the event with a talk about using SMRT Sequencing to detect clinically relevant structural variation. Introducing himself as a consumer of sequencing data, rather than a technology expert, he described a collaboration with PacBio to uncover structural variants associated with intellectual disability. Patients with this symptom often never receive a diagnosis, and while that situation has improved with exome and whole genome sequencing, it still isn’t fully addressed. In an assessment of 100 patients with severe disability, Brunner said, 38 of them still had no diagnosis even with WGS.
To overcome the challenge, his team turned to the Sequel System. In preliminary results from sequencing five trios, his team found 21 Mb of sequence revealed with SMRT Sequencing that had gone unresolved with short-read sequencing. They also found as many as 25,000 structural variants per genome, two-thirds of which were invisible to short-read technology. Brunner noted that the ability to phase data with PacBio sequencing provides “very useful information.” He also predicted that this approach could be implemented in the clinic within a year, based on the time it took to move exome sequencing toward patient care.
Expansion Sequence Variations Underlie Distinct Disease Phenotypes in SCA10
Next up was Karen McFarland, Research Assistant Professor at the University of Florida, who spoke about recent efforts to resolve repeat expansion regions associated with spinocerebellar ataxia type 10 (SCA10), a progressive neurodegenerative disorder. SCA10 is associated with a repeat expansion in the ATXN10 gene, ranging from nine repeats in unaffected individuals to as many as 4,500 in affected people. That’s as much as 22.5 kb of sequence, which has precluded thorough characterization of the region in the past. With SMRT Sequencing, though, McFarland and her team have not only successfully sequenced the region, but have also been able to accurately detect interruptions in the sea of ATTCT repeats that appear to have clinical consequence. Recently, they used the Cas9 enzyme to perform target capture of the region directly from genomic DNA of family members with different ataxia-related phenotypes.
Multi-platform Discovery of Haplotype-resolved Structural Variation in Human Genomes
Charles Lee, Scientific Director of The Jackson Laboratory for Genomic Medicine, gave the final user talk. He focused on a study from the Human Genome Structural Variation Consortium to assess many different technologies for discovering structural variants. The analysis of Yoruban, Han Chinese, and Puerto Rican trios showed that no single tool is currently capable of capturing the full range of human structural variants, which can be larger than a megabase. SMRT Sequencing, though, was found to dramatically increase the number of variants that could be detected and contributed to a seven-fold overall increase in structural variation discovery, he said. Among the variants he finds particularly interesting to pursue are inversions and mobile element insertions.
PacBio Applications Updates and Future Roadmap
Our CSO Jonas Korlach spoke as well, offering updates on structural variation, targeted sequencing and Iso-Seq analysis, as well as a look at future SMRT Sequencing developments. On the structural variation front, he noted that tools have evolved enough now that PacBio users can access a full SV detection workflow — including sequencing, read mapping, variant calling, and visualization — in the new SV Calling Software. Learn more about it or try our new project calculator on our new structural variation web page. For amplification-free target enrichment using the Cas9 protocol mentioned by McFarland, Korlach said that development is currently underway to adapt it for the Sequel System. If you’re attending ASHG, you can find out more at the CoLab session on Friday or check out the amp-free targeted sequencing web page.
Looking ahead, he told attendees that by the end of the year there will be a new accelerated protocol for library prep, a doubling of sequencing yield, and updated analysis tools in SMRT Link 5.1. Another 2-fold improvement is expected in 2018, and with the introduction of a new SMRT Cell with 8 million ZMWs (8-fold increase) in early 2019, a combined 30-fold capacity boost compared to current throughput will be achievable. These advances will help support the rising number of population studies that require an ever-increasing number of high-quality human genome assemblies.
We’ll be reporting on more from ASHG in the coming days, so stick with us!
The annual meeting of the American Society of Human Genetics kicked off with a splash yesterday in Orlando, Fla. The PacBio team was thrilled that the opening talks in the presidential address and plenary session included a significant focus on increasing diversity in genetic studies to better characterize underrepresented populations. Nancy Cox, ASHG president, highlighted a number of excellent efforts to address this but noted, “Compared with what we need, what we’ve done so far is really just a drop in the bucket.” As regular blog readers know, we work closely with groups around the world to build population-specific reference genomes and improve structural variant calling for all groups, so her message really resonated with us.
The other headline event for us yesterday was a joint workshop from the Genome Reference Consortium and Genome in a Bottle consortium, called “Getting the Most from the Reference Assembly and Reference Materials.” In the GRC portion of the event, talks from NCBI’s Valerie Schneider and McDonnell Genome Institute’s Tina Graves-Lindsay were particularly interesting for our team. Schneider offered a brief overview of GRC projects and a deeper dive into accomplishments and challenges in improving the human reference genome. As she noted, the latest build is GRCh38, which is a combination of sequences from some 70 people; she and her team continue to patch the assembly, correcting mistakes and adding new representations of genetic variation without changing existing coordinates. Graves-Lindsay spoke about the need to generate more high-quality human genome sequences for better characterization of people from all ancestries, and reported on efforts to do just that. The GRC has now used SMRT Sequencing, among other tools, to produce data for 10 diploid and two haploid genomes as a means of adding allelic diversity to the reference. Two chromosome-level assemblies reflect just how complete and contiguous these resources are. They plan to produce FALCON-unzip assemblies for all samples to increase quality and usefulness even further.
In its section of the workshop, GIAB covered reference materials, variant benchmarking, and more. Among the speakers, Baylor’s Fritz Sedlazeck and NIST’s Justin Zook gave talks that were especially meaningful for us. Sedlazeck, who has developed several important algorithms for working with long-read sequencing data and calling structural variants, encouraged the community to support large population studies focused on structural variation. He also announced a new project that will entail whole genome sequencing for 100 individuals using SMRT Sequencing and linked-read sequencing, with the goal of detecting and phasing structural variants. In his talk, Zook spoke about bringing the principles of metrology to genomics, and said that GIAB benchmarks are already widely used for clinical validation. His team is now focused on particularly challenging genetic regions and variants and recently released a structural variant call set.
The excitement continues today with our workshop, Population and Clinical Genetics Studies Using Long-read SMRT Sequencing. Please join us on the lower level of Hilton Orlando, Orange Ballroom D (connected to the Orange County Convention Center) at 12:30 p.m. for lunch and learning with Han Brunner, M.D., Charles Lee, Ph.D., FACMG, Karen McFarland, Ph.D., and our own Chief Scientific Officer Jonas Korlach, Ph.D.
Stay tuned for more updates as we report back daily on the great science being presented at ASHG.
The Human Genome Structural Variation Consortium, a successor to the 1000 Genomes Project Consortium, recently released a preprint describing an in-depth study of structural variant (SV) detection in human genomes. The scientists found that PacBio long-read sequencing and complementary technologies dramatically improve sensitivity for these important genomic elements when compared to standard short-read sequencing.
“Multi-platform discovery of haplotype-resolved structural variation in human genomes” comes from lead authors Mark Chaisson, Ashley Sanders, and Xuefang Zhao; along with corresponding authors Charles Lee, Evan Eichler, and Jan Korbel; and many other consortium members. The study involved extensive sequencing of three family trios — Han Chinese, Puerto Rican, and Yoruban — for comprehensive discovery of structural variants. “The Han Chinese and Yoruban Nigerian families were representative of low and high genetic diversity genomes, respectively, while the Puerto Rican family was chosen to represent an example of population admixture,” the scientists write.
To date, attempts to identify all structural variants in a human genome using short-read technology have been unsuccessful. These variants are biologically and clinically relevant, so it is imperative that the community find better ways of resolving them. As the authors stated, “The incomplete identification of structural variants from whole-genome sequencing data limits studies of human genetic diversity and disease association.” For this project, they add, “we integrated a suite of cutting-edge genomic technologies that, when used collectively, allow structural variants to be assessed in a near-complete, haplotype-aware manner in diploid genomes.” Tools included short-read sequencing, SMRT Sequencing, optical mapping, synthetic long reads, and single-cell/single-strand sequencing. The authors also applied multiple analysis algorithms for each type of data, further improving sensitivity.
The team identified in each genome more than 800,000 indel variants smaller than 50 bp and nearly 32,000 structural variants 50 bp or larger. That is “a sevenfold increase in structural variation compared to previous reports, including from the 1000 Genomes Project.” The authors also report the identification of “156 inversions per genome—most of which previously escaped detection—as well as large unbalanced chromosomal rearrangements.”
An evaluation of the contribution by technology showed that PacBio sequencing has a threefold increase in sensitivity for structural variants compared to Illumina sequencing, likely resulting “from better access to intermediate-sized SVs (50 bp to 500 bp) and improved sequence resolution of insertions across the SV size spectrum,” the team notes. “The long-read sequence data provided us with an unprecedented view of genetic variation in the human genome,” the scientists add. “Using ~15 kbp reads at an average of 40-fold sequence coverage per child, we have been able to span areas of the genome that were previously opaque and discover three to fourfold more structural variation when compared to short-read sequencing platforms.” They estimate that 77% of insertions are routinely missed by variant-calling algorithms based on short-read data.
“This study represents the most comprehensive assessment of structural variation in human genomes to date,” the authors write. “We predict that a move forward to full-spectrum SV detection using an integrated approach demonstrated in this study will increase the diagnostic yield in patients with genetic disease, SV-mediated mutation, and repeat expansions.”
This research will be showcased next week at the American Society of Human Genetics (ASHG) annual meeting in Orlando. Charles Lee will present a talk entitled “Multi-platform Discovery of Haplotype-resolved Structural Variation in Human Genomes” at the PacBio workshop on Wednesday, October 18th at 12:30 pm. In the afternoon, Xuefang Zhao will present poster #1501, “Comprehensive Discovery of Genomic Variation from the Integration of Multiple Sequencing and Discovering Technologies.” Check out the complete list of presentations at ASHG 2017 featuring SMRT Sequencing.
Good news for cloud-loving PacBio users: genomics analysis service Bluebee has now implemented HGAP4 for de novo assembly of SMRT Sequencing data. It creates a fully automated, end-to-end assembly pipeline for genomes of all sizes from unmapped BAM files. The pipeline is available now to all Bluebee users.
This new analysis service expands the use of SMRT Sequencing data beyond the realm of bioinformatics experts. Bluebee’s platform is designed for ease of use and alleviates the pressure of setting up compute clusters or other on-site tech solutions. Bluebee will also handle updating the pipeline as new versions are released, so users can just focus on their genome results.
The Bluebee de novo Assembly Pipeline for PacBio Sequencing is the exact implementation of the PacBio HGAP4 pipeline as provided by SMRT Link (v5.0.1), covering pre-assembly, assembly, and polishing steps. Users can adjust tool parameters if they wish to customize the pipeline.
Bluebee offers a private cloud service for genomics analysis that serves all major European countries and US cities, plus Canada and Asia Pacific. Based in the Netherlands, the company has implemented strict, multi-layered security controls and its platform is compliant with all applicable security and regulatory standards.
Attending the annual American Society of Human Genetics (ASHG) meeting next week? For a demonstration of the pipeline, stop by and visit during our expert hours:
PacBio booth #722 – Thursday, 2:30 – 3:30 PM
Bluebee booth #522 – Friday, 12:30 – 1:30 PM
Check out our full range of ASHG activities – we look forward to seeing you in Orlando!
A new preprint from scientists at the University of Guelph in Canada and the University of Pennsylvania reports the evaluation of SMRT Sequencing with the Sequel System as a replacement for Sanger platforms for amplicon sequencing. They found that long-read PacBio sequencing was highly accurate, exceeded Sanger coverage metrics, and reduced costs by 40-fold.
“A Sequel to Sanger: Amplicon Sequencing That Scales” comes from lead author Paul Hebert, senior author Evgeny Zakharov, and collaborators. The team embarked on this project in the hopes of finding a suitable amplicon sequencing alternative to costly Sanger technology. Short-read sequencers have not succeeded for this application because “the short read lengths and high error rates of most platforms constrain their utility for amplicon sequencing,” they note. “While recent studies have established that Illumina and Ion Torrent platforms can analyze 1 kb amplicons with good accuracy, their need to concatenate short reads creates risks to data quality linked to the recovery of chimeras and pseudogenes.” In addition, cost improvement of these platforms compared to Sanger is just three- to four-fold.
They turned to the Sequel System and circular consensus sequencing (CCS) of amplicons which were indexed with all combinations of 100 distinct forward and 100 distinct reverse primer barcodes. CCS covers the same amplicon several times in a single read to ensure high accuracy, followed by consensus calling of molecules with the same barcode pair. For a rigorous evaluation, the scientists simultaneously analyzed barcoded amplicons from the mitochondrial cytochrome c oxidase I gene from 10,000 separate DNA extracts and representing more than 5,000 Arthropoda species, in a single SMRT Cell. The PacBio system was thus tested with a range of previously difficult sequencing aspects, from homopolymers to varied GC content, evenness of coverage across isolates, and more. SMRT Sequencing results were compared to those from Sanger technology.
The study found that the Sequel System delivered excellent accuracy and that the technique was robust. “Across this range of templates, SMRT sequencing showed no points of failure,” the scientists report. “SMRT sequences also had a major advantage over their Sanger counterparts as they regularly provided complete coverage for the target amplicon.” Unidirectional Sanger reads, for example, were frequently truncated and bidirectional reads varied noticeably in length, generally reflecting homopolymer runs.
While this project focused on shorter amplicons, the team notes that Sanger technology has known limitations for templates longer than 1 kb because of the need to analyze overlapping amplicons. Even in CCS mode, SMRT Sequencing reads are long enough that multi-kilobase templates can easily be covered several times.
The team reports that sequencing capacity makes the Sequel System particularly attractive for this application. “Because it can characterize amplicon pools from 10,000 DNA extracts in a single run, the SEQUEL reduces costs 40-fold from Sanger analysis,” they write. “Exploitation of this capacity is aided by the fact that data processing is simple.” Unlike Sanger data, which calls for visual inspection of results, or short-read data, “SMRT sequences can be processed with an automated pipeline,” they add.
Today is International Ataxia Awareness Day (IAAD), and we’re proud to be participating in this worthy cause. Ataxia is a group of rare, degenerative neurological diseases with a number of different presentations; many involve muscle tremors, loss of motor skills, and difficulty walking. As many as 150,000 people in the United States have some form of ataxia.
Because there are so many different types of ataxia, one of the most important early steps for those affected is getting an accurate diagnosis. There are several hereditary ataxias, and genetic testing is increasingly useful for pinpointing the exact type affecting a patient.
An ongoing challenge for accurate genetic testing, however, is that some ataxias are associated with repeat expansions — making these genetic regions difficult to characterize with short-read or even Sanger sequencing technologies. Recently, scientists have made significant leaps forward by applying SMRT Sequencing. Work from Tetsuo Ashizawa from Houston Methodist Research Institute and his colleagues Birgitt Schuele and Karen McFarland from the Parkinson’s Institute and University of Florida has been particularly impressive. In 2015, they published results from sequencing the complete repeat expansion ranging from ~5.3 kb-7.0 kb in several patients, finding novel interruption motifs that may help explain a specific phenotype. Just this month, they reported using a Cas9 method to snip out the region of interest and long-read PacBio sequencing to characterize it, again speculating that the presence or absence of repeat interruptions has an impact on pathology. “Single molecule sequencing paired with SMRT/Cas9 capture approach allowed us to characterize the genetic composition of the complete repeat expansion which revealed a novel phenotype-genotype correlation for Parkinson’s disease and ATXN10,” Ashizawa and his collaborators wrote.
We congratulate Ashizawa and all the other scientists working so hard to improve outcomes for patients with ataxia. Like any rare disease community, these patients lack resources available to more common disease groups, such as robust patient/caregiver organizations and extensive research funding. On this special day, we ask that you help raise awareness for the people who live with ataxia, and the scientists focused on finding new treatments.
To participate in #IAAD17 and learn more about the symptoms, treatment, diagnosis and different types of ataxia, please visit the excellent website of the National Ataxia Foundation. To hear more about Ashizawa and Schuele’s work, check out this video:
We’re pleased to release a new data set along with an allele phasing GitHub software workflow for those interested in exploring SMRT Sequencing data from an Alzheimer’s disease candidate gene study. Our team collaborated with Integrated DNA Technologies (IDT) to design a 35-gene panel targeting candidate Alzheimer’s disease genes identified as potential genetic risk loci across many GWAS and linkage studies. Long-read PacBio sequencing was applied to brain and skeletal tissue from two individuals diagnosed with Alzheimer’s disease and a wide range of variants were detected, from SNPs to indels, and larger structural variations up to several kilobases in size. Additionally, alleles were successfully phased which provides a more comprehensive understanding of the biological significance of the variants present in the samples. Here’s an example screenshot of a BIN1 gene phased into two phase blocks across a 62,641 bp region:
The samples were sequenced using the Sequel System (Sequel Chemistry 1.2) and analyzed with our newly updated Phasing Consensus Analysis for Targeted Sequencing Data GitHub repository. Data sets and related files are available on our PacBio DevNet. Captures of 7 kb genomic fragments for brain and skeletal muscle tissues were each sequenced on a single SMRT Cell, yielding roughly 8 GB of mappable data to the human reference genome.
For more about this data collection, don’t miss the upcoming webinar, “Characterizing Alzheimer’s disease candidate genes and transcripts with targeted, long-read, single-molecule sequencing” hosted by IDT on Wednesday, September 27th. We will be deep diving into the project and illustrate how coupling genomic and transcriptomic captures with xGen® Lockdown® probes enable informative results and insights beyond SNPs.
Register now to attend at 7:00 am PDT/10:00 am EDT / or at 11:00 am PDT/2:00 pm EDT.
A new publication from scientists at The Rockefeller University and PacBio presents reference-grade, phased diploid genome assemblies for two important avian models for vocal learning, Anna’s hummingbird and zebra finch. Results are expected to help establish genome quality standards for the G10K and B10K sequencing projects, in addition to providing a better foundation for neuroscience studies.
Published in GigaScience, “De Novo PacBio long-read and phased avian genome assemblies correct and add to reference genes generated with intermediate and short reads” comes from lead author Jonas Korlach, senior author Erich Jarvis, and collaborators. The team undertook this project to improve the quality of genome assemblies available for these birds, demonstrating that key genes of interest were completely represented in single contigs. Existing assemblies produced with Sanger or short-read sequencing were incomplete and highly fragmented, precluding the comprehensive scientific view required for a deeper understanding of vocal learning.
By incorporating SMRT Sequencing, the team not only raised the bar for assembly quality but also phased the genomes using FALCON-Unzip, a diploid assembly tool. The new zebra finch assembly represented “a 108-fold reduction in the number of contigs and a 150-fold improvement in contiguity compared to the current Sanger-based reference,” the authors write. For hummingbird, the PacBio assembly led to “a 116-fold reduction in the number of contigs and a 201-fold improvement in contiguity over the reference.” Both assemblies had contig N50s greater than 5 Mb. “These long-read and phased assemblies corrected and resolved what we discovered to be numerous misassemblies in the references,” the scientists report, “including missing sequences in gaps, erroneous sequences flanking gaps, base call errors in difficult to sequence regions, complex repeat structure errors, and allelic differences between the two haplotypes.”
The team assessed gene content of the assemblies with CEGMA and BUSCO comparisons. In both cases, the number of complete or nearly complete genes increased. They also used RNA-seq to evaluate the reference genomes, finding that the PacBio long-read assemblies increased “total transcript read mappings compared to the Sanger-based reference … suggesting more genic regions available for read alignments,” they write.
Finally, the scientists conducted in-depth interrogations of four genes particularly important for vocal learning. EGR1, for instance, has gaps in previous zebra finch and hummingbird reference genomes. In both SMRT Sequencing assemblies, though, the gene was fully resolved and spanned in a complete contig. There were similar improvements for DUSP1, FOXP2, and SLIT1.
“We found that the long-read diploid assemblies resulted in major improvements in genome completeness and contiguity, and completely resolved the problems in all of our genes of interest,” the scientists report. “We now, for the first time, have complete and accurate assembled genes of interest that can be pursued further without the need to individually and arduously clone, sequence, and correct the assemblies one gene at a time.”
For more, check out our recent release of Iso-Seq data for hummingbird and zebra finch.
In a recent BMC Genomics paper, scientists in the Netherlands report a high-quality genome assembly for Folsomia candida, a soil-dwelling arthropod. The organism, which is known for reproducing parthenogenetically (and only when infected with Wolbachia), is frequently used in the lab for toxicity testing.
Lead author Anna Faddeeva-Vakhrusheva, senior author Dick Roelofs, and collaborators at Vrije Universiteit Amsterdam and other institutions describe their findings in “Coping with living in the soil: the genome of the parthenogenetic springtail Folsomia candida.” The team chose SMRT Sequencing to characterize the genome so they could learn more about the organism’s reproductive process and stress response.
F. candida has a diploid genome with seven pairs of chromosomes. The scientists generated a 221.7 Mb assembly with a contig N50 of 6.5 Mb. It is remarkably complete, with just 0.1% of all bases marked by gaps. Analysis revealed that repeat segments comprise more than 23% of the genome, and GC content was more than 37%. The team performed a number of quality-control and validation steps, concluding that assembly quality was excellent. The assembly also included the complete 15 kb F. candida mitochondrial genome.
The team was particularly interested in genome content acquired through horizontal gene transfer. A systematic analysis of all genes predicted by the assembly identified more than 800 acquired genes, most of which came from bacteria, fungi, and protists. The complement of horizontally transferred genes was impressive: “This number is among the highest found in metazoan genomes, being only exceeded in rotifers and some nematode species,” the scientists report.
Another highlight of the study came from a focus on F. candida’s endosymbiont Wolbachia. “Parthenogenesis is most likely imposed by Wolbachia,” the team writes. “The presence of Wolbachia is essential for reproduction: animals cured of Wolbachia by antibiotic treatment lay eggs that fail to hatch and develop.” The arthropod sequencing effort also yielded a complete assembly of the endosymbiont, which with its 1.8 Mb genome is the largest strain of Wolbachia ever discovered. Forty-eight genes were found to harbor ankyrin repeats, which are known for “mediating protein-protein and protein-DNA interactions with the host cells,” the scientists note.
Intriguingly, the team identified a functional antibiotic biosynthesis cluster, “suggesting the production of yet undiscovered antimicrobial compounds in an animal genome,” they conclude. “This high quality genome will be instrumental for evolutionary biologists investigating deep phylogenetic lineages among arthropods and will provide the basis for a more mechanistic understanding in soil ecology and ecotoxicology.”
A panel session at the recent Precision Medicine Leaders Summit, held in San Diego last month, offered great perspectives on the need to better represent global ethnic diversity in order to make the most of genomic advances for all patients.
Panelists included Robert Sebra from the Icahn School of Medicine at Mount Sinai; NCBI’s Valerie Schneider; Benedict Paten from the University of California, Santa Cruz – representing the Global Alliance for Genomics and Health; and Justin Zook, co-leader of the NIST Genome in a Bottle (GIAB) Consortium. The discussion was moderated by our own Luke Hickey.
The session kicked off with a look at a study published in the New England Journal of Medicine that found a greater number of incorrect genetic test results in black Americans than in white Americans for an inherited heart disorder. Along with other examples, that provided a good foundation for a conversation about the risk of health disparities based on genomic data. The speakers also discussed how the human reference genome and other sources contribute to genetic bias.
Clearly, benefits from precision medicine should be equally available to people from all ethnic groups. The panel talked about ongoing efforts to improve the human reference genome and other resources by including more ethnic diversity, as well as recent efforts to establish new population-specific reference genomes. Examples included GIAB projects to sequence trios, resulting in high-quality Ashkenazim Jewish ancestry genome assemblies, and international programs that have recently presented excellent assemblies for Korean, Chinese, Japanese, African, and Danish individuals.
Looking to the future, panelists spoke about improving study and test design to represent diversity. They also discussed how the community can work to make precision medicine more accurate for all ethnic groups, including data-sharing programs and more.
A compelling new paper from scientists at the Parkinson’s Institute and Clinical Center, Houston Methodist Research Institute, and several other organizations demonstrates the importance of fully sequencing repeat expansion regions for a clearer understanding of the underlying biology of the diseases they cause. This publication also offers a look at how CRISPR/Cas9 capture can be used in combination with SMRT Sequencing to access the expanded repetitive region at a base level resolution without any PCR bias.
“Parkinson’s disease associated with pure ATXN10 repeat expansion” comes from lead authors Birgitt Schüle and Karen McFarland, senior author Tetsuo Ashizawa, and collaborators. The study involved a Mexican family with one individual previously diagnosed with Parkinson’s disease and several members with spinocerebellar ataxia.
Clinical genetic testing had found an ataxia-associated pentanucleotide repeat expansion in the patient with Parkinson’s, and this team hoped to learn more. “To further genetically characterize the ATXN10 repeat expansion and to better understand the phenotypic differences of progressive cerebellar ataxia with seizures and parkinsonism,” they write, “we employed several advanced and novel molecular genetic techniques to dissect the genetic structure of the repeat expansion in this family.”
Among those techniques was a new method that combined the sequence-specific endonuclease activity of the CRISPR/Cas9 system with long-read SMRT Sequencing. The team reports that they were able to use this method to snip out genomic ATXN10 repeat expansion regions, some spanning up to 7 kb in length, and sequence them “as one continuous fragment without prior amplification of the genomic DNA.” This was done for six family members, with results indicating that most affected family members had a string of 480 ATTCT repeats followed by about 920 ATTCC repeat interruptions. Strikingly, the family member with ataxia and parkinsonism had a different expansion: more than 1,300 ATTCT repeats but no ATTCC repeats. “We propose that the absence of repeat interruptions play a role in the underlying disease process acting as a genetic modifier and leading to the clinical presentation of L-Dopa responsive parkinsonism,” the scientists write, adding that the repeat interruptions may contribute to the development of epilepsy.
“Single molecule sequencing paired with SMRT/Cas9 capture approach allowed us to characterize the genetic composition of the complete repeat expansion which revealed a novel phenotype-genotype correlation for Parkinson’s disease and ATXN10,” the team adds, highlighting the importance of adding to existing knowledge of repeat expansion types and possible phenotypes. “We conclude that the underlying genetic architecture of ATXN10 repeat expansions is critical for presentation of clinical phenotypes and presumably also the underlying pathology.”
A recent paper in the journal Angewandte Chemie describes using SMRT Sequencing to characterize biosynthesis of a psychotropic product in Psilocybe carpophores, better known as magic mushrooms. Scientists from the Hans Knöll Institute in Germany report that the work could pave the way to synthetic production for pharmaceutical use.
“Enzymatic Synthesis of Psilocybin” comes from Janis Fricke, Felix Blei, and Dirk Hoffmeister. The team aimed to uncover the enzymatic mechanisms of biosynthesis for psilocybin, culminating in the characterization of four related enzymes: PsiD, PsiK, PsiM, and PsiH. “In a combined PsiD/PsiK/PsiM reaction, psilocybin was synthesized enzymatically in a step-economic route from 4-hydroxy-l-tryptophan,” the authors write.
Scientists used PacBio sequencing to analyze Psilocybe cyanescens, resulting in a 61.3 Mb assembly with just 217 contigs (meanwhile, a short-read assembly of a closely related mushroom for the same project required more than 2,900 contigs to represent just 41.3 Mb). After identifying the genes involved in producing psilocybin, the team validated the work by splicing them into E. coli and confirming the biosynthesis event.
Since its structure was first characterized in 1959, scientists have been seeking ways to synthesize psilocybin — but without success. As the study authors note, their new results finally “may lay the foundation for its biotechnological production.”
In an article from Chemical & Engineering News, the University of Minnesota’s Courtney Aldrich said the discovery will be important “for developing a fermentation process for production of this powerful psychedelic fungal drug.”
If you’re interested in avian vocal learning or want to explore a PacBio Iso-Seq data set generated with the Sequel System, we have good news. We’ve just released data from Iso-Seq interrogations of brain tissue from two avian models of vocal learning, Anna’s hummingbird (Calypte anna) and zebra finch (Taeniopygia guttata), sequenced in collaboration with the Erich Jarvis and Olivier Fedrigo labs at the Rockefeller University.
If you’re not familiar with the Iso-Seq method, it’s the long-read sequencing answer to short-read RNA-seq studies. By using SMRT Sequencing for a transcriptome project, scientists can generate full-length isoform data, clearly capturing alternative splicing events to see the real diversity of transcripts. Unlike RNA-seq approaches, the Iso-Seq method takes advantage of long-read data to fully span transcript isoforms from the 5’ end to their poly-A tails, eliminating the need for error-prone transcript reconstruction and inference processes. With the Sequel System, Iso-Seq projects are low cost and time efficient. Currently we recommend only 1-2 SMRT Cells per tissue type for genome annotation.
For this data set, we used the Iso-Seq method to characterize the transcriptomes of two birds, with brain total RNA. The two species’ brain samples were barcoded, pooled, and sequenced using 4 SMRT Cells on the Sequel System. An average of ~460,000 reads was generated per SMRT Cell; total sequencing data yields ranged from 6.1 to 7.7 Gb per SMRT Cell. More than 15,000 isoforms were identified in each species, including thousands that had not been previously annotated in each bird and 400 to 500 new genes.
The data set contains both the raw pooled sequences and the processed, demultiplexed sequence files, separated by species and excluding any raw sequences not containing barcodes. Our initial analysis of these data is presented in this poster (Vierra et al.), which is being presented this week at the Genome 10K and Genome Science Conference at the Earlham Institute. It demonstrates how improved loading on the Sequel System simplifies library prep and how both command-line and new SMRT Link tools can be used for analysis. It also illustrates how full-length transcript data can help identify additional exons and UTRs.
Enjoy the data!
If you use the data and our analyses in our publication before we complete our study, please cite:
Michelle N. Vierra, Sarah B. Kingan , Elizabeth Tseng , Tyson Clark, Ting Hon, William J. Rowell, Jacquelyn Mountcastle, Olivier Fedrigo, Erich D. Jarvis, Jonas Korlach. From RNA to Full-Length Transcripts: The PacBio Iso-Seq Method for Transcriptome Analysis and Genome Annotation. Genome10K and Genome Science Conference Abstracts 2017.
A new paper in Scientific Reports presents results from a transcriptome analysis for Oryctolagus cuniculus. The work was done with SMRT Sequencing, which allowed scientists to discover novel transcripts and increase the diversity of known transcripts for the rabbit.
“A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing” comes from lead authors Shi-Yi Chen and Feilong Deng, senior author Song-Jia Lai, and collaborators at Sichuan Agricultural University. In the paper, the scientists note that an ongoing challenge in rabbit studies has been the dearth of gene-level data. “Most of the existing gene models are just derived from in silico prediction with lack of the reliable annotation on alternative isoforms and untranslated regions,” they write.
The team turned to SMRT Sequencing to generate full-length transcripts and avoid the well-known assembly pitfalls of short-read transcript data. “We employ this technology to sequence polyadenylated RNAs of rabbit and provide a transcriptome-wide landscape in relation to gene models and alternative isoforms,” they report.
The scientists pooled and sequenced RNA samples from several organs and tissues collected from three New Zealand white rabbits. After filtering, they were left with more than 36,000 high-confidence transcripts from nearly 15,000 genes. That included quite a bit of novel information: “more than 23% of genic loci and 66% of isoforms have not been annotated yet within the current reference genome,” the scientists write. Their interest in alternative splicing was rewarded as well, with the final transcriptome containing nearly 25,000 alternative splicing events and more than 11,000 alternative polyadenylation events. Those numbers represent an order of magnitude more alternative splicing than was characterized in the reference gene models. The project also turned up a significant amount of non-coding RNAs, represented by 17% of transcripts.
The scientists followed up on these findings with several validation studies, including an analysis of genes in the major histocompatibility complex. Their analysis demonstrates “the obviously improved power of PacBio transcripts for recovering the highly homologous sequences among ten MHC genes than the assembled transcripts from short reads,” they report.
According to the paper, scientists achieved their mission of more thoroughly characterizing the rabbit transcriptome. “The length distribution of the most 5′ exons of our PacBio transcripts is consistent with former report in human,” they write, “which would indicate the comparable sequencing completeness in rabbit.”
We’re looking forward to the discussion of many more vertebrate species’ genomes at the upcoming Genome 10K and Genome Science Conference 2017 hosted this week by The Earlham Institute.
Sniffles and NGMLR, structural variant detection and alignment algorithms developed in the Schatz lab for long-read sequence data, are already familiar to many in the PacBio community. Now, a preprint is available so users can see how these open-source tools perform in a variety of conditions.
“Accurate detection of complex structural variations using single molecule sequencing” comes from lead author Fritz Sedlazeck at Baylor College of Medicine, senior author Michael Schatz at Johns Hopkins University, and collaborators. The team notes that long-read sequencing has introduced a much more comprehensive means of discovering structural variants, many of which are missed by short-read sequence data. To take advantage of that capability, the scientists developed NGMLR, “a fast and accurate aligner for long reads,” and Sniffles, which “successively scans within and between the alignments to identify all types of [structural variants],” according to the paper. Sniffles is unique in its ability to routinely detect nested variants.
The scientists describe evaluating the performance of these tools for structural variant discovery using data from several different sequencing platforms. The tools were tested on data from a breast cancer genome, healthy human genomes, and Arabidopsis. Using a simulated human data set, the team found that NGMLR and Sniffles outperformed other algorithms such as BWA-MEM and PBHoney, detecting nearly 95% of structural variants with no false discoveries. While more than 94% of variants called by PacBio were confirmed by other platforms, the scientists report that “Oxford Nanopore had substantially worse concordance. … This systematic bias for deletions in the Oxford Nanopore data is most likely an error in the base calling.”
Sedlazeck et al. also found a concerning trend in structural variant calls when using short-read data. The authors note, “Using the short-read approach we detect, on average, 27 times more translocation events compared to using Sniffles within presumably healthy human data sets,” they note. An investigation into this phenomenon determined that mis-mapping of short reads in low-complexity regions leads to insertions being misidentified as translocations. “Overall, we could rule out 1,869 (83.18%) of the Illumina-based translocation calls as false,” they report.
Finally, the scientists assessed how much coverage is necessary to see the full picture of structural variation. For a healthy human genome, 15-fold coverage of SMRT Sequencing “has a precision of ~80% and recall of 69.64%,” they write. Boosting that to 30-fold coverage achieved similar comepleteness for the much more complex cancer genome. “This translates to a potential price reduction of several tens of thousands of dollars per sample,” they add. “These requirements will be reduced even more in the years to come as the throughput and read length increase and sequencing error rates decrease.”
“The versatility of these methods enables an unprecedented view into structural variations in the human genome and other genomes from long read single molecule sequencing data,” the scientists write. They predict that these and related improvements “will usher in a new era of high quality genome sequences for a broad range of research and clinical applications, and lead to new insights into polymorphic variation, pathogenic conditions, and the forces of evolution.”
Scientists from the University of Hong Kong recently reported results of a head-to-head comparison of long-read and short-read platforms for sequencing and assembly of a bacterial genome. They determined that only SMRT Sequencing was capable of generating highly accurate, complete assemblies. “Completing bacterial genomes should no longer be regarded as a luxury, but rather as a cost-effective necessity,” the team reports.
“PacBio But Not Illumina Technology Can Achieve Fast, Accurate and Complete Closure of the High GC, Complex Burkholderia pseudomallei Two-Chromosome Genome” was published in Frontiers in Microbiology by lead author Jade Teng, senior author Patrick Woo, and collaborators. For this project, scientists compared performance of the PacBio RS II Sequencing System with the Illumina HiSeq 1500. Their target was Burkholderia pseudomallei, which has at least 68% GC content as well as “highly repetitive regions and substantial genomic diversity,” the authors report.
After sequencing, the team attempted both hybrid and single-source assemblies. Working with Illumina data alone “resulted in a draft genome with more than 200 contigs,” they note, pointing out that the platform’s reliance on PCR amplification is inherently problematic for GC-rich genomes. Three different short-read assemblers were not able to improve results. The hybrid assembly of both sequencers’ data was also “not successful,” producing 74 contigs, the team reports.
Assembling only PacBio data, which was generated from a single SMRT Cell, led to a very different result. The approach “achieved complete closure of this two-chromosome B. pseudomallei genome without additional costly bench work and further sequencing, demonstrating its utility in the complete sequencing of bacterial genomes, particularly those that are well-known to be difficult-to-sequence,” the scientists write. The chromosome contigs of the assembly aligned to the organism’s reference genome with better than 99.9% accuracy. Importantly, the assembly accurately characterized “the number of CDSs and their distributions in each subsystem, four ribosomal operons, the highest number of core and virulence proteins (coverage of query protein sequence and amino acid identity ≥80%), and MLST gene loci,” the team adds.
The Illumina assembly, on the other hand, was unable to resolve these elements. “Extraordinarily high coverage of Illumina reads were observed in several collapsed repeat regions, including regions containing varying copies of mobile element proteins and ribosomal operon,” Teng et al. report. “We reasoned that Illumina sequencing was not able to resolve these repeat regions as their sequence reads were not long enough to span different kinds of repeats with unique flanking sequences.”
The scientists also included an assessment of project cost. “To completely sequence a bacterial genome using Sanger sequencing or the second generation sequencing platforms, the main bulk of the cost, labor and time is spent in the gap-filling phase,” they write. “It has been estimated that when using these second generation sequencing platforms, around 95% of the money and time are spent in completing the last 1% of the bacterial genome.” But the calculation is very different for SMRT Sequencing. “Although the cost per base is more expensive for the PacBio RS II platform compared to short-read sequencing technology, no additional manual work after de novo assembly is required,” the team concludes, “and the benefit of obtaining an accurate number of individual replicons and an intact assembly of repetitive regions and mobile genetic elements justify the initial cost.”
Earlier this year, scientists from Korea reported results from a transcriptome study of Pacific abalone. In this paper, the team used SMRT Sequencing to demonstrate that alternative splicing and gene expression have sex-specific signatures in these organisms.
“Alternative Splicing Profile and Sex-Preferential Gene Expression in the Female and Male Pacific Abalone Haliotis discus hannai” comes from lead authors Mi Ae Kim and Jae-Sung Rhee, senior author Young Chang Sohn, and collaborators. They focused on abalone, a marine gastropod, because of its importance to Korean aquaculture: the species they studied is estimated to represent about 10,000 metric tons of production each year.
As H. discus hannai has grown in economic importance, new genomics resources have become available, including a genetic linkage map and some RNA-seq data. This latest project was designed to glean more information about abalone to provide a clearer view of its biological function. Scientists chose to focus on sex-specific transcriptomes, using the Iso-Seq method to study gene content in male and female members of the species. They analyzed several tissue types — including gonads, muscle, gills, and more — and defined 15,110 protein-coding genes in females and 12,145 in males. Of those, 519 genes in female and 391 genes in male produced alternatively spliced transcripts.
To validate these findings, the team investigated expression profiles for six genes known to be sex-preferential in related organisms. Two of the three female-specific genes and all three male-specific genes were highly expressed in their respective samples. “Taken together, these studies strongly suggest the intactness of the sex-specific isoform DB of the Pacific abalone,” the authors write.
“The information obtained in this study represents the first significant contribution to sex-specific genomic resources, as well as isoform information,” the scientists conclude. “These data will provide an essential genomic reference that could be used for further diverse genetics- and physiology-based research using abalones.”
If you weren’t at the 36th International Society for Animal Genetics Conference in Dublin, you missed more than a chance to drink Guinness and practice an Irish brogue. The PacBio team had a great time at ISAG, learning about the latest in animal science and updating attendees on the advantages of SMRT Sequencing for generating high-quality genome assemblies and annotations.
The conference drew more than 750 scientists from around the world, and we were truly impressed by the quality of research they presented in talks and posters. Long-read PacBio sequencing is already making a difference for scientists in this community, many of whom are focused on improved breeding programs. Genome assemblies powered by SMRT Sequencing were presented for many economically important species, including chicken, sheep, goat, pig, cattle, horse, camel, and Atlantic herring. There were also several presentations featuring PacBio long-read sequencing data for immune region haplotypes such as the leukocyte receptor complex and the major histocompatibility complex.
We hosted a morning seminar that demonstrated how SMRT Sequencing provides comprehensive views of animal genomes and transcriptomes. John Williams from the University of Adelaide presented a preliminary assembly of the water buffalo genome generated with Sequel System data. A member of the International Buffalo Genome Consortium, Williams described limitations in contiguity and completion for previous sequencing efforts that used short-read data for this important livestock animal. Seeking a reference-grade assembly, the scientists turned to PacBio long reads, using FALCON-Unzip to phase more than half of the diploid genome. Though the assembly is not yet polished, Williams reported a stellar contig N50 of 18.7 Mb. The other seminar speaker was our own Emily Hatas, who discussed the chicken genome annotation generated by Richard Kuo at the Roslin Institute. In that project, scientists used the Iso-Seq method with SMRT Sequencing to identify 64,000 transcripts, including more than 17,000 long non-coding RNAs that had not been previously annotated.
As sponsors of the event, we also had fun encouraging attendees to snap creative photos with the toy animals we gave away at our booth. Check out the variety of clever snapshots!
We were delighted to be back at the University of Maryland this summer for our annual East Coast User Group Meeting. The day-long event, preceded by half-day workshops on sample prep and bioinformatics, exceeded our expectations. From the packed session hall to the terrific science and great discussions, the UGM facilitated the exchange of best practices and new suggestions for optimizing SMRT Sequencing performance for a variety of applications. Below is a recap of the day’s highlights, with several of the presentations available to download.
PacBio scientist Aaron Wenger presented the Structural Variant Calling application that is included in the SMRT Link v5.0 software release. The application utilizes the read aligner NGM-LR, and features both a command-line tool called pbsv and a web interface. Noting that most of the genetic difference between any two people lies in structural variation, he showed that short-read sequencers cannot detect the vast majority of these important variants. Wenger demonstrated that even low-coverage SMRT Sequencing can be used to discover structural variants; in an experiment, 10-fold coverage revealed almost 100% of homozygous variants and nearly 90% of heterozygous variants in a human individual.
Michael Schatz from Johns Hopkins University gave a talk entitled “In Pursuit of Perfect Genome Sequencing” in which he walked through three key metrics for evaluating genome quality: correctness (basepair accuracy), completeness (no gaps in the sequence), and contiguity (sequence ordered as on the physical chromosomes). Schatz compared the leading sequencing technologies available today, and explained that PacBio SMRT Sequencing is the most capable technology for all three metrics.
Continuing the human genome theme, Ricardo Mouro Pinto from Massachusetts General Hospital spoke about using SMRT Sequencing to quantify CAG repeat instability in Huntington’s disease. Caused by a CAG repeat expansion, Huntington’s occurs when a person’s genome harbors 40 or more copies. Pinto noted that typically, the longer the repeat, the younger the person is at disease onset. The Huntington’s locus is difficult to enrich because it is resistant to PCR amplification. By using Cas9 digestion to perform non-amplification-based target enrichment followed by PacBio sequencing, Pinto’s team was able to capture wild type and disease alleles with no amplification bias. He noted that results are preliminary, and he hopes to expand the number of samples studied to get a better handle on CAG instability.
Representing the plant community, Hamid Ashrafi and Hamed Bostan from North Carolina State University tag-teamed a presentation on the blueberry genome and transcriptome. The fruit plant naturally occurs in diploid, tetraploid, and hexaploid genomes. The scientists generated a high-quality diploid assembly using SMRT Sequencing and noted that long reads were essential to get through the highly repetitive genome. Next, they used Iso-Seq to study several types of tissue from diploid, tetraploid, and hexaploid blueberry plants, finding many transcripts missed by short-read sequence data. Using both genome and transcriptome approaches was particularly important, Ashrafi noted, because SNPs explain only a small portion of natural variation for this plant, and he believes that alternative splicing and structural variants likely contribute a much larger proportion of variation. The team is still analyzing results but said that switching to long reads was “a dream come true.”
On the microbial front, Jethro Johnson from The Jackson Laboratory for Genomic Medicine gave a talk on full-length 16S rRNA sequencing, which is useful for taxa identification. By genotyping or using short-read data, Johnson said, so much of the information in the variable regions of 16S is missed that it often is impossible to accurately classify organisms. So, Johnson turned to SMRT Sequencing and circular consensus sequencing (CCS), which generates highly accurate long reads. Johnson applied CCS for a mock bacterial community of 36 species and found that SMRT Sequencing offered accurate results for identification. In studies of fecal samples, PacBio sequencing was able to provide a unique identification in cases where short-read sequencing generated ambiguous results. The team is now expanding SMRT Sequencing results to include internal transcribed spacer regions.
In a separate presentation, Phillip Tai from the University of Massachusetts Medical School highlighted the use of long-read sequencing for genome population sequencing of adeno-associated viruses. These harmless viruses have gained new interest recently as a vector for gene therapies, so Tai’s lab is interested in analyzing large groups of them to filter out any that would not be ideal vectors. By applying SMRT Sequencing to recombinant AAVs, they generate complete resolution of the vector genome, including the difficult-to-sequence inverted terminal repeats. This accomplishment could have tremendous value in the gene therapy field, he said.
The meeting also included some new tools and protocols from the community. New England Biolabs’ Bo Yan presented SMRT-cappable-seq, a method for characterizing operons across an entire bacterial genome. It involves capping the 5’ end of bacterial primary transcripts and using SMRT Sequencing to produce full-length transcripts. Yan said the protocol increases library prep efficiency and accurately defines and links the transcription start site and transcription termination site (something short reads cannot do). A validation project in E. coli revealed 840 novel operons, extending 40% of annotated operons in RegulonDB. In another talk, Manuel Tardaguila from the University of Florida discussed SQANTI, a new tool to perform quality control for long-read transcripts. The pipeline performs classification, curation, and quantification of transcripts to filter out any artifacts and ensure that scientists analyze only the highest-quality results. SQANTI incorporates PacBio data, a reference genome, and other resources to conduct its rigorous evaluation.
We’d like to thank our hosts for the meeting, the Genomics Resource Center, Institute for Genome Sciences at the University of Maryland, as well as our partners: Advanced Analytical Technologies, Diagenode, and Sage Science. And, of course, thanks to all the scientists who took time out of their busy schedules to make this event a success!