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星期二, 2018年8月7日

PacBio Sequencing Reveals Food Processing & Pathogenic Strains of Yeast are the Same Species

Candida krusei, a form of yeast that is known to be drug-resistant and able to cause opportunistic infections in humans

What’s in a name? Too much, when it comes to the taxology of yeast, it turns out.

Scientists from University College of Dublin have found that two distinctly named species of yeast are in fact 99.6% identical at the base pair level, and collinear. In other words, they are the same species.

It was a bit of a shock, especially considering one of the yeast species, Pichia kudriavzevii, is commonly used in food production and classified by the US FDA as “generally recognized as safe,” while the other, Candida krusei, is known to be drug-resistant and able to cause opportunistic infections in humans.

“The existence of multiple names for this species has almost certainly impeded research into it,” the researchers write. “We suggest that P. kudriavzevii should be the only name used in future.”

Their study, published in PLOS Pathogens, highlights the importance of gathering comprehensive genetic data of organisms.

The Irish team, led by Kenneth H. Wolfe and first author Alexander P. Douglass, is the first to sequence the type strain of C. krusei. Genome sequences had been published previously for four P. kudriavzevii strains and one C. krusei clinical isolate, but they were highly fragmented, and none of them provided a chromosome-level assembly or transcriptome-based annotation.

The researchers produced high-quality reference genomes for a C. krusei type strain called CBS573 and the CBS5147 type strain for P. kudriavzevii. They then annotated the genomes with the help of RNA sequence data for CBS573, uncovering more than 5,100 protein-coding genes. They also re-sequenced 30 additional clinical and environmental isolates to explore the relationships between the strains and their genomic diversity.

Not only did the comprehensive assemblies clarify the genome content and structure, they uncovered some unexpected features of the genomes.

“One of the most unexpected features of the genome is the structure of its centromeres, which consist of a simple but large IR. The 99% DNA sequence identity of the 8–14 kb units that form the IRs means that centromere organization would have been difficult to deduce without long-read PacBio data.”

The data also allowed them to take a deeper dive into a question that has been perplexing scientists in the field concerning the sexual cycle of the yeast.

When P. kudriavzevii was first described, it was reported to be able to sporulate, forming one spore per ascus, but later studies reported that the type strain of P. kudriavzevii does not mate or sporulate.

“Our discovery that this strain is triploid provides a possible explanation for its failure to sporulate, or at least its failure to produce viable spores,” the authors write.

As for implications to health and safety, the authors say the yeast should no longer be used in food processing, as it “presents a potential hazard to the health of immunocompromised workers, and potentially also to consumers.”

They suggest that the closely related, non-pathogenic Pichia species be considered as possible alternatives for some industrial applications.

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星期三, 2018年8月1日

Something to Crow About: SMRT Sequencing Aids Conservation of Rare Hawaiian Bird

AlalaBrought to the brink of extinction, the future of Hawaii’s only lineage of the crow family (Corvidae) is looking up thanks to intensive conservation genomics efforts using PacBio de novo assemblies.

In Hawaiian mythology, the ‘alalā is said to lead souls to their final resting place on the cliffs of Ka Lae, the southernmost tip on the Big Island of Hawaii. As one of the largest native bird populations, it also had a vital role in the ecosystem, helping to disperse and germinate seeds of many indigenous plant species.

Disease, predators and shrinking habitats led to a complete loss of the species in the wild. A captive breeding program led by San Diego Zoo Global managed to save nine ‘alalā and has successfully bred around 140 more to date. But the captive birds also face challenges, including low hatching success and signs of poor genetic diversity due to inbreeding, with the majority of the population linked to a single founding pair.

Not satisfied with following family trees to determine suitable mating pairs, a research team from the San Diego Zoo Institute for Conservation Research, the University of Hawaii, and other organizations produced a high-quality genome assembly based on SMRT Sequencing. The team believed a comprehensive genome assembly could provide a more detailed picture of population-level genomic diversity and genetic load of Corvus hawaiiensis, as well as more accurate estimates of molecular relatedness to guide breeding decisions. And they were right.

Led by Jolene Sutton, assistant professor at the University of Hawaii, Hilo, the team created an assembly which has provided critical insights into inbreeding and disease susceptibility. They found that the ‘alalā genome is substantially more homozygous compared with more outbred species, and created annotations for a subset of immunity genes that are likely to be important for conservation applications.

As reported in the latest issue of Genes — and featured on its cover — the quality of the assembly places it amongst the very best avian genomes assembled to date, comparable to intensively studied model systems.

“Such genome-level data offer unprecedented precision to examine the causes and genetic consequences of population declines, and to apply these results to conservation management,” the authors state. “Although pair selection and managed breeding using the pedigree has kept the inbreeding level of the ‘alalā population at a relatively low level over the past 20 years, the intensive and ongoing conservation management of the species requires a more detailed approach.”

Since the generation of the ‘alalā assembly, several projects have been initiated that rely heavily on use of the new resource, the authors state. To better understand the impact of population bottlenecks over the past 100 years, and to provide a clearer picture of how much diversity can likely be maintained into the future, the team is using targeted SNP-capture to compare genomic diversity in museum and modern ‘alalā, for example. Plans are also underway to genotype every individual ‘alalā against this new reference to further inform the choice of breeding pairs in captivity as well as the management of an ‘alalā release project started in 2017.

“Genomic data derived from our analyses are an essential component of the current and future recovery of the ‘alalā,” the authors write. “As the size of both the captive and wild ‘alalā populations continue to increase, the integration of genomic data as part of the conservation management effort will help to maximize the genetic health of the species well into the future.”


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星期一, 2018年7月30日

Diversity of Unexpected CRISPR Edits Revealed by SMRT Sequencing

Image by Ernesto del Aguila III, National Human Genome Research Institute, NIH

A new Nature Biotechnology publication is sending reverberations through the CRISPR and gene therapy communities. The discovery that the widely used CRISPR/Cas9 method results in far more genomic changes than previously thought — including big deletions and rearrangements — was made possible by the use of long-read SMRT Sequencing.

Repair of double-strand breaks induced by CRISPR–Cas9 leads to large deletions and complex rearrangements” comes from Michael Kosicki, Kärt Tomberg, and Allan Bradley at the Wellcome Sanger Institute. The scientists aimed to better understand the possible universe of on-target edits (rather than the better-studied off-target effects) made in a controlled environment, starting with a 5.7 kb amplicon from the X-linked PigA locus in mouse embryonic stem cells. “Thus far, exploration of Cas9-induced genetic alterations has been limited to the immediate vicinity of the target site and distal off-target sequences, leading to the conclusion that CRISPR–Cas9 was reasonably specific,” they write.

Their findings led to a collective groan among CRISPR scientists and the businesses based on this technology. “We report significant on-target mutagenesis, such as large deletions and more complex genomic rearrangements at the targeted sites in mouse embryonic stem cells, mouse hematopoietic progenitors and a human differentiated cell line,” Kosicki et al. report. “We speculate that current assessments may have missed a substantial proportion of potential genotypes generated by on-target Cas9 cutting and repair, some of which may have potential pathogenic consequences following somatic editing of large populations of mitotically active cells.”

The heterogeneous nature of DNA repair after CRISPR edits was previously observed by Gasperini et al. which shared the strategy of long read SMRT sequencing to get a more clear picture of editing outcomes. In both cases, choosing long-read SMRT Sequencing allowed a larger region adjacent to the intended edit site to be surveyed, uncovering unexpected changes caused by CRISPR-Cas9 cuts. A number of these changes would have been impossible to spot with short-read sequencing, such as large edits deleting an adjacent primer binding site that would have been used to check the region. “The most frequent lesions in these cells were deletions extending many kilobases up- or downstream, away from the exon,” the scientists note. “We conclude that, in most cases, loss of PigA expression was likely caused by loss of the exon, rather than damage to intronic regulatory elements.” In one case, the team even found a de novo insertion — “a perfect match to four consecutive exons derived from the Hmgn1 gene” — that they believe came from spliced, reverse-transcribed RNA.

These sweeping edits weren’t the only bad news in the paper. The scientists repeated the original experiment four times to determine whether the same edits would be seen each time and found that they were not. “Each biological replicate differed substantially, despite a large number of unique deletion events sampled, indicating that the diversity of potential deletion outcomes is vast,” they report.

The CRISPR method has been considered quite promising as a gene-editing tool to cure disease, and this publication does not suggest that the authors’ findings would necessarily derail that idea. Instead, they urge others in the field to be more comprehensive in analyzing genomes before and after the use of CRISPR for a clearer view of its effects. “Results reported here … illustrate a need to thoroughly examine the genome when editing is conducted ex vivo,” they conclude. “As genetic damage is frequent, extensive and undetectable by the short-range PCR assays that are commonly used, comprehensive genomic analysis is warranted to identify cells with normal genomes before patient administration.”

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星期五, 2018年7月27日

Deep Dives into DNA of Marine Biology

What lies beneath? Photo by Ruben Gutierrez.

“Live every week like it’s Shark Week,” 30 Rock character Tracy Jordan once quipped to Kenneth the Page, referencing the week-long, dorsal-finned programming phenomenon that has become the Discovery Channel summer ratings mainstay.

If it involves diving deeply into the science of the maligned species, we’re all in favor. But why stop there?

On our companion long-form Medium blog, we hosted our own Marine Week to highlight recent scientific discoveries across the seas.

  • In “Healthy Marine Ecosystems Rely on Their Tiniest Inhabitants,” we explore how the health of ocean habitats relies on more than the activities of our finned friends. Just as human health is proving to be linked to the microbial communities in our guts, marine health is influenced by the bacteria in its ecosystems. A group of Thai scientists are studying the marine microbiology of coral reefs in the Gulf of Thailand and the Andaman Sea to glean the role bacteria might play in the health of the habitat and its responses to environmental stressors, such as elevated seawater temperature.
  • The orange clownfish, Amphiprion percula, may have been immortalized in the comedic film “Finding Nemo,” but its importance to the scientific community is no joke. In “Finding Nemo’s Genes: International Team Creates First Reference Genome of Orange Clownfish,” we visit an effort led by Tim Ravasi of King Abdullah University of Science and Technology in Saudi Arabia and Phil Munday of James Cook University in Australia, to create molecular resources for one of the most important species for studying the ecology and evolution of coral reef fishes, as well as a model species for social organization, sex change, mutualism, habitat selection, lifespan, and predator-prey interactions.
  • Aquaculture has become an increasingly important source of sustainable seafood. And similar to the city singles scene, its viability has a lot to do with sex. In “Deep in the Dating Pool,” we look at how studies into sex differentiation of two marine species — Nile tilapia (Oreochromis niloticus) and abalone (Haliotis discus hannai)– can help commercial and conservation breeding efforts. Long-read sequencing and the Iso-Seq method were key to the success of these efforts by two international research groups.
  • In “A Fish Tale: Tracing the Divergence of a Species,” we explore what it takes for one species to evolve into another, with medaka as the model. A popular pet since the 17th century because of its hardiness and pleasant coloration, scientists are more interested in the genetics of the medaka, but earlier attempts to sequence the fish’s 800 Mb genome were not the best quality, and had 97,933 gaps in their sequence. So researchers at the University of Tokyo started from scratch, using Single Molecule, Real-Time (SMRT) Sequencing. This advanced technology allowed them to study difficult-to-detect centromeres and changes in DNA structure that were missing in the previous genome assemblies.

Hungry for more? Head over to bioRxiv, where a team of Japanese and American researchers, led by Shawn Burgess at the NIH’s National Human Genome Research Institute, have reported on the assembly of the goldfish (Carassius auratus) genome and the evolution of its genes after whole genome duplication. As a very close relative of the common carp (Cyprinus carpio), goldfish share the recent genome duplication that occurred approximately 14-16 million years ago in their common ancestor, and the combination of centuries of breeding and a wide array of interesting body morphologies “is an exciting opportunity to link genotype to phenotype as well as understanding the dynamics of genome evolution and speciation,” the authors state.

Generating a high-quality draft sequence of a “Wakin” goldfish using 71-fold coverage PacBio long-reads, the team identified 70,324 coding genes and more than 11,000 non-coding transcripts and found that that two sub-genomes in goldfish retained extensive synteny and collinearity between goldfish and zebrafish. However, “ohnologous” genes were lost quickly after the carp whole-genome duplication, and the expression of 30% of the retained duplicated gene diverged significantly across seven tissues sampled.

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星期二, 2018年7月24日

SMRT Science, Tips & Tricks Presented at Leiden Meeting

SMRT cell jewelry

SMRT Art: Jewelry created from upcycled SMRT cells by Olga Pettersson.

When was the last time you sent your DNA off to a day at the spa? Olga Pettersson of the SciLifeLab at Uppsala University lets her molecules relax for up to a week at room temperature to enable them to untangle, achieve better chemical purity, and better sequencing output.

It was one of many practical pointers shared by presenters at the popular three-day gathering of PacBio users in Leiden, Netherlands last month. SMRT Leiden featured the scientific discoveries and analytical achievements of more than 30 speakers.

Inge Kjaerbolling of the Technical University of Denmark shared her tricks using the new Aspergillus genomes for linking compounds to metabolite clusters. Zev Kronenberg, whose name recently graced Science for the cover story on the great apes comparative genome, discussed some of the tools he has developed in his new role as a Phase Genomics scientist: Polar Star for breaking chimeric PacBio contigs using Hi-C; Matlock for Hi-C data pre-processing; and FALCON-Phase, a method for using Hi-C to scaffold FALCON-Unzipped PacBio genomes.

Day 1 also featured several scientific talks about large genome projects, including: the Bat1K initiative from Sonja Vernes of the Max Planck Institute; the genome sequencing of the Zika carrier, the Aedes aegypti mosquito, from Rockefeller University’s Ben Matthews; the tomato genome project, from Mohamed Zouine of INRA/INP Toulouse; and the maize genome from Doreen Ware of USDA/Cold Spring Harbor, who prophesied: “The next green revolution will be data driven.”

Day 2 kicked off with a densely packed and awe-inspiring keynote talk by Shinichi Morishita of the University of Tokyo, covering topics with implications for human disease, speciation, structural variants, haplotype phasing, and metagenomics. It was followed by a talk from Laurence Ettwiller of New England Biolabs on a new full-length transcriptome protocol for bacteria, as well as a preview of the forthcoming version of structural variation calling in PacBio’s official SMRT Link/SMRT Analysis software suite, by PacBio scientist Armin Töpfer.

Human disease was the topic of several other presentations. Stuart Scott from the Icahn School of Medicine in New York explained how he uses SMRT Sequencing to identify and phase variants important for human disease mutations. Marjolein Weerts from Erasmus MC, Netherlands, presented her work on inferring cancer signatures on the basis of low-frequency mitochondrial DNA (mtDNA) circulating in the blood stream. And Birgitt Schuele of the Parkinson’s Institute discussed her latest publication that applied PacBio’s No-Amp method to sequence repeat expansions in the ATXN10 gene.

Dutch scientists Alex Hoischen and Yahya Anvar discussed additional applications in human genetics and precision medicine, and Martin Pollard of the Sanger Institute delved into population genomics, describing an effort to generate an expanded reference panel of MHC haplotypes from African populations.

Sergey delivered the Day 3 keynote on trio binning.

The third day of the event was the SMRT Informatics Developers Conference, which featured a mixture of bioinformatics talks and open discussion. Speakers went into depth about de novo assembly, structural variation, amplicon sequencing, and PacBio’s Iso-Seq method for sequencing full-length RNA transcripts.

Sergey Koren’s talk about TrioBinning, an new approach for complete haplotype reconstruction, was especially popular, and David Heller (Max Planck) illustrated his graph-based approach, SVIM, for calling structural variants using long reads.


For in-depth coverage of the event, check out the four-part Medium series by PacBio Scientist Liz Tseng:

Read More »

星期二, 2018年7月17日

Long Look Into Ant Brains Provides Epigenetic Insights

To understand the epigenetic regulation of brain function and behavior, scientists are turning to ants. To understand the ants, they are applying the accurate, long reads of SMRT Sequencing.

While the genetic code of many types of ant have been combed through thanks to several genomes assembled through whole-genome shotgun sequencing, there have only been brief glimpses and guesses regarding gene regulation. Existing assemblies are highly fragmented drafts, making epigenetic studies nearly impossible.

Ant brainsEager to determine the epigenetic changes responsible for phenotypic and behavioral plasticity in Camponotus floridanus and Harpegnathos saltator ant species, a team of researchers from the Epigenetics Institute of the University of Pennsylvania’s Perelman School of Medicine used SMRT Sequencing to de novo assemble the two genomes, which had been previously sequenced using short reads.

Improved genome continuity led to comprehensive annotations of both protein-coding and non-coding RNAs, and answered some questions about the differential gene expression that allows some worker ants to become acting queens in their colonies.

In a paper published in Cell Reports, first author Emily J. Shields, lead author Roberto Bonasio and their collaborators described how they solved some mechanistic mysteries through PacBio long-read sequencing.

Harpegnathos worker ants are characterized by their unique reproductive and brain plasticity that, in the absence of a queen, allows some of them to transition to a queen-like phenotypic status called “gamergate,” which is accompanied by major changes in brain gene expression.

Previous work by the group in Harpegnathos and in the more conventional Florida carpenter ant Camponotus floridanus had suggested that epigenetic pathways, including those that control histone modifications and DNA methylation, might be responsible for differential deployment of caste-specific traits; pharmacological and molecular manipulation of histone acetylation has been shown to affect caste-specific behavior in Camponotus ants, suggesting a direct role for epigenetics in their social behavior.

“Although the molecular mechanisms by which environmental and developmental cues are converted into epigenetic information on chromatin remain subject of intense investigation, it has become clear that non-coding RNAs play an important role in mediating this flow of information,” the authors write.

In particular, they were interested in long non-coding RNAs (lncRNAs), which are transcripts longer than 200 base pairs that are not translated into proteins. Annotated extensively in human, mouse, bees, zebrafish, Drosophila melanogaster and Caenorhabditis elegans, no comprehensive annotation of lncRNAs in ants has been reported, limiting the reach of ant species as model organisms.

As many cis regulatory and epigenomic mechanisms take place at short-to-medium range (10–100 kb), the scientists wanted to span large repetitive regions and create longer gap-free regions of sequence (i.e., longer contigs) than those produced by previous short-read assemblies, so they turned to PacBio.

“Long PacBio reads allowed us to assemble across longer repeats than previously possible, greatly improving the contiguity of the Harpegnathos and Camponotus genomes,” the authors write.

They sequenced genomic DNA isolated from Harpegnathos and Camponotus workers using SMRT Sequencing, obtaining a sequence coverage of 70-fold for Harpegnathos and 53-fold for Camponotus, with longer contigs (on average more than 30-fold larger than a prior 2010 assembly) and scaffolds with fewer gaps. The assemblies have scaffold N50 sizes larger than 1 Mb, and gaps smaller than in all other insect genomes available on NCBI at the time of writing.

The UPenn team annotated protein-coding genes using a combination of methods, and they discovered more than 300 high-confidence lncRNAs, several of which displayed developmental-, brain-, or caste-specific expression patterns, suggesting important roles in development and brain function.

They were also able to identify some biologically relevant genes missing in the older versions of the genome assemblies. Most notably, a Gp-9-like gene previously unannotated in the Harpegnathos genome was found to be differentially expressed in worker brains compared to gamergates. Mass spectrometry analyses identified two peptides mapping exactly to the newly predicted sequence, confirming the accuracy of the updated gene model.

“This gene was not previously detected as differentially expressed, likely because its closest homolog in the old annotation contains many sequence disparities, reducing the RNA-seq coverage mapped to this gene in both castes,” the authors write.

The UPenn team will use the new assemblies to direct their future explorations of neuroepigenetics in ants. They also hope the work will have a wider impact on the field.

“Our greatly improved Harpegnathos and Camponotus assemblies deliver several critical benefits to further development of these ant species into molecular model organisms,” the authors conclude. “These improvements… will lead to greater understanding of the genetic and epigenetic factors that underlie the behavior of these social insects.”

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星期五, 2018年7月13日

Egyptian Rousette Bat Genome Provides Clues to Antiviral Mystery

When humans are infected with the Marburg virus, the result is often lethal, with hemorrhagic fever and other symptoms similar to Ebola. When bats are infected, the result is…. nothing. The tiny mammals remain asymptomatic.

In order to crack this antiviral mystery, a multi-institutional team of scientists sequenced, assembled and analyzed the genome of the bat species Rousettus aegyptiacus, a natural reservoir of Marburg virus and the only known reservoir for any filovirus.

Their findings contradicted previous hypotheses about bat antiviral immunity, which assumed that bats had enhanced antiviral defenses, controlling viral replication early in infection, and developing effective adaptive immune responses as a result. The new analysis suggests that an inhibitory immune state may exist instead.

Led by Boston University researchers Thomas B. Kepler and Stephanie S. Pavlovich, with Gustavo Palacios of the United States Army Research Institute of Infectious Diseases and others from Columbia University, the University of Nebraska, the NIH’s National Center for Biotechnology Information, and the Viral Special Pathogens Branch of the Centers for Disease Control and Prevention, the study in Cell describes several differences between immune responses in bats and humans.

Among them was an expanded and diversified KLRC/KLRD family of natural killer cell receptors, MHC class I genes, and type I interferons in the bats, which dramatically differ from their functional counterparts in other mammals.

“Such concerted evolution of key components of bat immunity is strongly suggestive of novel modes of antiviral defense,” the authors write.

The stark difference between bat and primate antiviral responses has long motivated scientists to characterize the genes involved in the immune system of bats, but previous efforts relied on genomes generated with low-coverage sequencing or with only short-read sequencing technologies. Such assemblies limit the ability to resolve repetitive regions of the genome where important immune gene loci reside, the authors note. So they turned to PacBio long-read sequencing, combined with paired-end short-reads, to generate a high-quality annotated genome for the Egyptian rousette bat. The result is a 1.91 Gb Raegyp2.0 genome, the most contiguous bat genome available.

Natural Killers

Bat genome

They used the genome to study two large classes of immune genes: natural killer (NK) cell receptors and type I interferons (IFNs). Previous studies have reported the absence of canonical NK cell receptors in bat genomes, and others have suggested that significant differences exist in type I IFNs between bats and humans. Diving deeper, the new study found an unusual expansion of the KLRC (NKG2) and KLRD (CD94) gene families in R. aegyptiacus relative to other species, “showing genomic evidence of unique features and expression of these receptors that may result in a net inhibitory balance within bat NK cells.”

“The expansion of NK cell receptors is matched by an expansion of potential MHC class I ligands, which are distributed both within and, surprisingly, outside the canonical MHC loci,” the authors note.

They also observed that the type I IFN locus is considerably expanded and diversified in R. aegyptiacus, with members of the IFN-u subfamily being induced after viral infection and showing antiviral activity.

“All these features strengthen the notion of the unique biology of bats and suggest the existence of a distinct immunomodulatory mechanism used to control viral infection,” the authors conclude.

“Our findings are consistent with the hypothesis that certain key components of the immune system in bats have coevolved with viruses toward a state of respective tolerance and avirulence, although tolerance is likely not the only mechanism at play.”

The team notes that definitive tests of their hypotheses may be possible with the development of further experimental reagents for cytometry and biochemical intervention, and that such reagents are being developed now with information made available by the completed genome project.

And while the genome for R. aegyptiacus is providing useful information about how bats resist viral infections, it is just one species of interest to scientists who would like to better understand the genetics of bats in order to shed light on human and ecological biology. The Bat1K initiative is an effort by more than 140 scientists around the world to decode the genomes of all 1,300 species of bats using SMRT Sequencing and other technologies.

Read More »

星期三, 2018年7月11日

Maize Collaborators Embark on Ambitious 26-line Pangenome Project

Computational biologist Doreen Ware harvests maize tissue for RNA isolation. Photo by Miriam Chua c/o USDA

The first reference genome for maize variety B73, completed in 2009, was a major milestone, and an improved version released by Cold Spring Harbor Laboratory scientists in 2017 provided a deeper dive into the genetics of the complex crop. Yet even this new robust reference is not enough for Kelly Dawe, Doreen Ware and Matt Hufford, who have taken up another ambitious project: creating a 26-line pangenome reference collection in just two years.

“Maize is not only an important crop, but an important study species for answering basic questions about how plants grow and adapt to different environments,” says Ware, a computational biologist at USDA and Cold Spring Harbor Laboratory.

Interestingly, the genome differs significantly between individuals. A study comparing genome segments associated with kernel color from two inbred lines revealed that 12 percent of the gene content was not shared – that’s much more diversity within the species than between humans and chimpanzees, which exhibit more than 98 percent sequence similarity. The new project will create multiple reference genomes to reflect this diversity.

“By relying on a single type specimen as the sequence reference for most of the genetic information in maize, we may be missing much of the highly valuable natural variation in maize,” Ware says.

Beyond B73, the most extensively researched maize lines are the core set of 25 inbreds known as the NAM founder lines, which represent a broad cross section of modern maize diversity. SMRT Sequencing and BioNano optical mapping, which were essential in the creation of the groundbreaking 2017 B73 maize reference, will be used in the new $2.8 million National Science Foundation-funded project led by Dawe at the University of Georgia. They will create comprehensive, high-quality assemblies of these 25 inbreds, plus an additional line containing abnormal chromosome 10.

Plant genomes are notoriously difficult to sequence, and maize is particularly challenging because the vast majority of its 2.3 Gb diploid genome — a staggering 85 percent — is made up of highly repetitive transposable elements that other types of sequencing can’t address. Understanding these regulatory and structural elements is crucial to modern breeding efforts that aim to improve productivity across marginal environments and under changing climate.

“The sequenced lines will include varieties from both tropical and temperate regions, and their sequences should help us understand how corn has adapted to these different environments,” said Hufford, a co-principal investigator on the project and assistant professor at Iowa State University. “Understanding the ways corn adapts can facilitate development of lines for novel conditions.”

PacBio Sequencing will be essential as the team assesses the role of structural variation such as presence-absence and copy number variation in the determination of agronomic traits, Ware says.

The assemblies, along with information about the genes and their expression patterns, will be cataloged and made available to the public through her Gramene.org data resource.

“To go from a single reference to a broad perspective on the entire genetic repertoire of genes and gene expression patterns will be a major step forward in how we approach genome analysis in crops,” said Dawe, Distinguished Research Professor in UGA’s Franklin College of Arts and Sciences department of genetics and principal investigator on the grant. “It’s something that has not happened for any crop at this scale.”

Read about Doreen Ware’s original comprehensive maize genome project and about efforts at Corteva Agriscience™, Agriculture Division of DowDupont™ (formerly DuPont Pioneer) to create their own multiple maize reference library.


Read More »

星期二, 2018年7月10日

One Million Genomes Meeting Discusses Progress and Promise of Population-Scale Genomics

Keystone Symposia logo


The PacBio team was honored to attend an excellent Keystone Symposium in Hannover, Germany recently. The event, “One Million Genomes: From Discovery to Health,” offered a rare look at large-scale human genome projects, with many top-notch speakers.

The meeting featured speakers from many national genomics efforts, including China, Estonia, Israel, the UK, and the US. Each of these individual national efforts is essential to overcome the representation bias seen in human genome databases today. Underrepresented groups are currently less likely to get actionable results from clinical genetic tests, a situation that threatens to confer the benefits of precision medicine disproportionately to people of European ancestry. Many of the new population projects have incorporated SMRT Sequencing, either to produce a reference-grade de novo assembly or to generate structural variation data about participants of diverse ancestry.

Genome Asia 100k logo

A highlight of the meeting for us was the closing talk from Jeong-Sun Seo of Seoul National University Bundang Hospital and Macrogen in South Korea. Professor Seo discussed the GenomeAsia 100K Project and Asian reference genomes. Seo reported on three de novo, reference-grade Asian genomes – Chinese HX1, Japanese JRGv1, and Korean AK1 – all generated with SMRT Sequencing. These genomes enable more accurate re-sequencing of 4.5 billion Asian people, which Seo explained is useful to detect medically relevant variants in this population. At the time of publication, the AK1 genome was the most contiguous personal human genome ever reported, with a contig N50 over 18 Mb.

Seo also presented initial results from a new project that is using PacBio sequencing to detect structural variants in 300 Mongolian individuals. He observed that more of the structural variants in AK1 were detected with PacBio sequencing of the first 30 Mongolian individuals than had been seen in 2,504 individuals from the 1000 Genomes Project, which relied on short-read sequencing. This likely reflects two factors: Asian-specific variation and the greatly increased sensitivity of PacBio sequencing for structural variants.

Also at the event, PacBio scientist Ralph Vogelsang presented a poster about population-scale discovery of structural variants that showed how SMRT Sequencing is uniquely suited to detecting large and often complex variants, which are known to cause disease but are frequently missed by short-read sequencing approaches. The poster also includes a helpful map of ongoing population-focused genome projects.

Congratulations to all of the scientists around the world contributing to these important efforts. We look forward to seeing the many new discoveries they enable!

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星期三, 2018年6月27日

Plant and Animal SMRT Grant Will Enable Insight Into Sightless Fish

Cave barbatulaWhen German diver Joachim Kreiselmaier reached the deepest parts of the Danube-Aach cave system, he couldn’t believe his eyes: a “strange fish,” with a pale body coloration and smaller eyes and larger nares and barbels than the loaches typically spotted nearby. He had discovered the first cavefish in Europe, and the northernmost in the world.

“This is spectacular, as it was believed that the Pleistocene glaciations prevented fish from colonizing subterranean habitats north of 41° latitude,” said ecologist Jasminca Behrmann-Godel of the Limnological Institute of the University of Konstanz, who examined the fish Kreiselmaier brought back to the surface. “Initial genetic studies, together with knowledge on the geological history of the region, indicate that the cave loach population is amazingly young — not older than 20,000 years.”

The mysteries of the new species, Cave barbatula, will now be investigated by Professor Dr. Arne Nolte of the University of Oldenburg, Germany, and Assistant Professor Dr. Fritz Sedlazeck from the Human Genome Sequencing Center at Baylor College of Medicine in Houston, Texas, as part of the 2018 Plant and Animal SMRT Grant.

The grant will provide Nolte and Sedlazeck with access to the PacBio Sequel System at GENEWIZ, as well as the materials needed and bioinformatics support to conduct comparative genomic sequencing on the newly discovered European cavefish.

“This grant enables us to establish the genome assembly of the European cavefish and identify genetic variants from its surface-water ancestors. We are fascinated by changes in the sensory system and pale pigmentation of the fish and we will compare its genomic makeup with the Mexican cavefish which is an important model organism in developmental biology,” Sedlazeck said. “The outcome of this study will enable us to understand the initial steps that lead to the evolution of cave animals and impact our understanding of how multiple phenotypes evolve among vertebrates.”

“The combination of PacBio’s powerful genomics platforms and GENEWIZ’s depth of experience in DNA and next-generation sequencing provides researchers like Drs. Nolte and Sedlazeck the technology and support they require to further their discoveries and understanding of the world around us,” added Dr. Ginger Zhou, Vice President of Global Next Generation Sequencing for GENEWIZ.

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星期二, 2018年6月26日

It’s a Small World After All at ASM Microbe 2018

ASM Microbe attendees at the PacBio booth

When it comes to bacteria, resistance is not always futile, or so we learned at the annual meeting of the American Society for Microbiology. One of our favorite events of the year, ASM Microbe was full of fun puns, giant pathogen dolls, and amazing science spanning basic molecular biology and physiology, antimicrobial agents and resistance, environment, ecology and evolution, and clinical and public health microbiology.

We invited attendees to get hands on at our booth — quite literally — and our giant interactive hands art piece was a big hit. Four of our Certified Service Providers — The University of Maryland Institute for Genome Sciences, GENEWIZ, Macrogen, and RTL Genomics — were also on hand to answer questions about our technology, which was further highlighted in a presentation by Principal Scientist Cheryl Heiner on “Single Chromosomal Genome Assemblies on the Sequel System with Circulomics High Molecular Weight DNA Extraction for Microbes,

Microbial multiplexing was a hot topic, and we were excited to share information about new tools we recently released to make it easier and less expensive to sequence microbial genomes on the Sequel System. The streamlined workflow — from library preparation to genome assembly — includes the release of two new 8-plex barcoded adapter kits specifically validated for multiplexing microbial genomes, a multiplexing calculator to ensure even coverage when pooling barcoded samples, streamlined de-multiplexing with SMRT Link v5.1.0, and optimized setting for microbial genome assembly with HGAP4.

Advances enabled by PacBio long-read sequencing technology were also showcased in more than 25 posters and presentations. Several posters focused on bioinformatics methods. Lee Katz’s cleverly titled “Kraken with Kalamari: Contamination Detection” poster attracted a lot of interest. Katz, a scientist with the CDC, described how leveraging a curated database of closed PacBio genomes with Kalamari enables better identification of bacteria when using the popular Kraken metagenomics tool for foodborne disease surveillance. Seok-Hwan Yoon of the Chun Lab presented another bioinformatics focused poster about the EzBioCloud project, a valuable taxonomy resource that has incorporated a staggering amount of PacBio data.

Other posters delved into understanding the evolution of human pathogens. CDC scientist Michael Weigand shared his findings about whooping cough resurgence in the United States in his talk, “Chromosome Rearrangement, Gene Amplification, and Insertion Sequence Elements in the Genome Evolution of Bordetella pertussis and the Genus Bordetella.” If you have an ASM Microbe login, check out a poster presentation of his work. Bordetella (of the birdie variety) was also the topic of an award-winning abstract, “Clonal Evolution and Genomic Diversification of Bordetella Hinzii in An Immunocompromised Host,” by Adrien Launay of the NIH during a rapid-fire presentation, “Microbe, Know Thy Host.” Three students from the University of New Hampshire working in the lab of Cheryl Whistler presented work on the biology and ecology of Vibrio. Sarah Eggert and Jillian Means investigated the the pathogenesis of vibrio parahaemolyticus, the leading cause of seafood-borne bacterial infections, and the spread of this pathogen into the Gulf of Maine. Jennifer Calawa won an Outstanding Abstract Award for her work on the comparative genomics of two closely related Vibrio fischeri strains with varying symbiotic capabilities. Attendees also learned more about the amazing new resource released by the UK’s National Collection of Type Cultures (NCTC), in partnership with the Wellcome Sanger Institute and PacBio: reference genome assemblies of 3,000 strains of important historic and modern bacteria, including some of the deadliest.

Of course, research interests at ASM microbe extend well beyond infectious disease. Anne Hatmaker of Tennessee’s Oak Ridge National Lab explored the potential of Megasphaera elsdenii — a bacterium found in the rumen of cattle — in the production of biofuels in her late-breaking abstract.

Finally, we used the meeting to launch what has also become an annual tradition: The Microbial Genomics SMRT Grant Program, made possible this year with the help of the University of Maryland’s Institute for Genome Sciences. You, too, can apply for the chance to win free SMRT Sequencing and bioinformatics analysis by submitting a 250-word proposal by July 20.

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星期四, 2018年6月14日

For Reference-Grade Human Genome Assemblies, SMRT Sequencing Yields Optimal Results

SMRT Sequencing is a go-to technology for generating reference-grade human genome assemblies, according to speakers in a recent webinar. In their presentations, Tina Graves-Lindsay from Washington University and Adam Ameur from Uppsala University spoke about diploid assemblies, discovering novel sequence, improving diversity of the current human reference genome, and much more. Finally, our own Paul Peluso gave a presentation that included the technology roadmap showing the next several upgrades for the Sequel System.

Graves-Lindsay began with efforts from the Genome Reference Consortium to “represent the full range of genetic diversity in humans,” a task requiring the generation of many population-specific references. She presented data from two haploid and 13 diploid genomes produced so far, and noted that two others are underway. For each reference, the scientists generate ~60-fold WGS coverage with PacBio, then assemble with FALCON. To assist with assembly QC and scaffolding, they merge the resulting sequence contigs with data from orthogonal long-range technologies such as Bionano Genomics or 10x Genomics. The approach has yielded impressive results: three of the 13 reference genomes achieved chromosome-level assembly; the highest contig N50 reached 26 Mb. To highlight the value of population-specific reference genomes, Graves-Lindsay offered some examples of regions that are not yet represented in the current human reference (GRCh38 build) – such as a 65 kb insertion found in a Yoruban assembly. To further resolve the diploid genome assemblies, her team is running FALCON-Unzip to generate haplotype-resolved contigs. These haplotigs better represent each of the maternal and paternal haplotypes for each genome, as opposed to a single collapsed contig sequence, and will serve as an allele-specific reference for the populations they represent.

Ameur’s talk focused on an effort that came out of SweGen, a population sequencing effort that covered 1,000 individuals in Sweden. His team chose two participants — one male and one female — and used SMRT Sequencing to produce reference-grade assemblies for each. They generated 75-fold WGS coverage for each individual, and combined PacBio assembled contigs with Bionano optical maps to produce highly contiguous genomes. By comparing results to the initial SweGen results, Ameur found that a large proportion of the 20,000 structural variants detected in each reference assembly were missed by short-read sequencing. The new assemblies also included a total of 24 Mb of novel genome sequence, not represented in GRCh38; the vast majority of that data came from repetitive regions 5 kb or longer. While about 30% of the novel sequence had no hits in NCBI, the nearly 70% remaining did match existing sequences, leading Ameur to suspect that at least some of those sequences had been mis-annotated because they were not found in the human reference. Now, his team is going back to the original SweGen short-read WGS data and aligning it against the new reference genomes, which is helping to improve variant detection in the Swedish population, resolve false-positive SNPs, and improve alignment in some coding regions.

The webinar’s final presentation came from Peluso, who offered a quick overview of the features of SMRT Sequencing and its growing use for high-quality assemblies. Of the 65 human assemblies most recently submitted to NCBI, 90% of those with a contig N50 greater than 1 Mb were generated with PacBio data. Ongoing population studies and reference genome projects aim to use SMRT Sequencing on more than 2,400 human genomes globally. Peluso also presented data from the recent effort to sequence a Puerto Rican genome, HG00733, which used the latest advances for the Sequel System (v2.1 chemistry and 5.1 software). The SMRTbell Express Template Prep Kit allowed for faster sample prep and better yield, leading to libraries that generated more than 50% of data in reads longer than 33 kb and a contig N50 of 31.4 Mb. Average output per SMRT Cell was 10 Gb. The new assembly compared favorably to the Sanger-assembled GRCh38.p12, with fewer contigs (982 vs. 1536) and only slightly smaller contig N50 (31.4 Mb vs. 56.4 Mb). Peluso described cost efficiencies using the latest Sequel System improvements for de novo assembly, noting that “the original human reference genome cost $3 billion, and today you can characterize a single human genome with PacBio for around $3,000 (1/1-millionth the cost), and build a reference-quality genome de novo for around $20,000.”

Peluso also announced the availability of FALCON-Phase, an improved phasing assembly tool that incorporates long-range Hi-C data and can be found on Github. Looking ahead, he said that simplified library prep is on the roadmap for midyear, with a chemistry update to improve accuracy and yield slated for release in late 2018. Next year, a new SMRT Cell 8M is expected to expand yield and reduce costs significantly.

The event concluded with an audience Q&A covering details about alignment stringency, shared structural variants across the Swedish population, decoy sequences, and more. If you missed the live webinar, watch the recording any time.

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星期二, 2018年6月12日

5 Ways to Attract More Customers to Your Sequencing Services

Many people who run a sequencing core lab would prefer to focus on science instead of business, but all core lab managers know that it’s imperative to keep a steady stream of clients and projects filling the pipeline. Here, we offer a handful of tips to help you expand your user base.


  1. Be fast, high-quality, and easy to understand

To you a queue for sequencing may look like you’re at the top of your game with high demand, but to customers it can be frustrating. Regularly updating processes to improve pipeline efficiency will ensure that your customers are getting the fastest service possible so they can complete their research. And if your lab is consistently backlogged, it may be time to consider expanding your capacity.

Related to efficiency, the quality of the product you put out is one of the surest ways to gain happy customers and repeat business and to prevent customers from expressing negative thoughts about your services. Remember the adage that a happy customer will tell two potential prospects about their experience, but an unhappy customer will tell ten. The PacBio technical support team and your local FAS are available via web or phone to help troubleshoot or train on a particular application.

In addition to having an efficient pipeline producing excellent customer data, it’s important to have a mechanism to report easy-to-digest results. Think about the high-level metrics your customers need to understand their results and provide that in a concise report when you deliver their data.

  1. Differentiate yourself

Your customers need to know why they should choose you from other service providers. Whether it be by application (de novo assembly, Iso-Seq analysis, targeted sequencing, etc.), by organism type (plant, animal, microbial, etc.), or by additional services (HMW DNA isolation, bioinformatics, etc.), own what you are good at and shout it from the rooftops.

  1. Focus on solutions, not workflows

You, as a service provider, are intimately aware of workflow details because they are essential to your day-to-day operations. However, your customers care about the solutions your workflows and results make possible. Tell prospects about the cool and meaningful science that your services have enabled. Case studies or publication feeds on your webpage are a great way to distribute this information.

  1. Don’t be afraid to learn new things

Maybe you’re a seasoned pro at generating large libraries for de novo assembly projects and you’ve been curious about providing a long-read RNA sequencing solution. Contact your local FAS and set up a training session! There’s no better way to show that you keep up with the latest and greatest advancements in sequencing technology than by regularly updating your services to reflect the most up-to-date applications of SMRT Sequencing.

  1. Marketing, marketing, marketing

It may seem obvious to some, but getting the word out about your services is a surefire way to get more interest — and ultimately more projects — into your pipeline. Contrary to popular belief, it doesn’t take a marketing consulting firm or an executive with a decade of experience to get started. From free things like using social media to highlight successful projects and promotional pricing, to low-cost events such as hosting webinars with core facility advocates as guest speakers, and all the way to paid ads and automated email campaigns, there are many ways to get the word out about your services at any budget level.


We hope this list was helpful! We will be posting on each of these tips in more depth throughout the rest of the year. Don’t miss out on any of them by subscribing to our blog.

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星期一, 2018年6月11日

New High-Resolution Genome Assemblies Expand Our Understanding of Human-Ape Differences

Ever since researchers sequenced the chimpanzee genome in 2005, they have known that humans share the vast majority of our DNA sequence with chimps, making them our closest living relatives. So what, exactly, sets us apart?

While prior ape genome assemblies were helpful in finding single nucleotide changes, many researchers speculate that a variation type that is more difficult to resolve, structural differences in regulatory DNA or in the copy number of gene families, play important roles in species adaptation. Large-scale efforts to sequence and assemble more ape genomes over the last 13 years have expanded our knowledge, but many structural variations (SVs) that distinguish the great apes remain unresolved. Additionally, the currently available draft ape genome assemblies, which contain tens to hundreds of thousands of gaps, are often compared against the much higher-quality human genome reference, introducing bias that “humanizes” the ape assemblies.

Now, an effort led by scientists at the University of Washington has closed most of those gaps by producing ab initio chimpanzee and orangutan genome assemblies where most genes are complete and novel gene models are identified.

Great ape genomes are improved using PacBio sequencingIn a recently published Science paper, first author Zev N. Kronenberg of the UW Genome Sciences department and presently at Phase Genomics, lead author Evan E. Eichler, of UW and the Howard Hughes Medical Institute along with a multi-institutional team describe how they coupled PacBio long-read sequence assembly and Iso-Seq cDNA sequencing with a multi-platform scaffolding approach to characterize lineage-specific and shared great ape genetic variation ranging from single base-pair to megabase-sized variants.

The team sequenced four genomes—two human, one chimpanzee and one orangutan—to high depth (>65-fold coverage) using SMRT Sequencing data, and generated ~3 Gb assemblies for each species where the majority of the euchromatic DNA mapped to <1,000 large contigs. They then scaffolded the chimpanzee and orangutan genomes without guidance from the human reference genome. By using the same exact methods for assembly, these ape genomes along with the Eichler group’s long-read assembly of the gorilla genome could finally be compared to one another and the human genome on a more level playing field.

“Recent advances in sequencing and mapping technologies now make more detailed investigations possible, not only of individual species but also entire clades of species,” the authors write. “We generated new great ape genome assemblies displaying improved sequence contiguity by orders of magnitude, leading to a more comprehensive understanding of the evolution of structural variation.”

Comparing these new high quality genome assemblies to 86 recently sequenced great ape genomes and a diverse set of human genomes from the Simons Genome Diversity Panel, they identified 17,789 fixed human-specific structural variants, including 11,897 human-specific insertions and 5,892 human-specific deletions. These figures double the number of predicted genic and putative regulatory changes that emerged in humans since divergence from nonhuman apes. Among this set, they focused on SVs that potentially disrupt genes or regulatory sequence, identifying 1,145 human-specific SVs with potential functional effects.

“Unbiased genome scaffolding led to the discovery of novel and more complex subcytogenetic differences between human and other great ape chromosomes that were previously missed,” the authors write. “Projecting these onto the human genome shows potential hotspots of structural variation by size or number of events.”

Among the discoveries were fixed human-specific structural variants enriched near genes that are downregulated in human compared to chimpanzee cerebral organoids, particularly in cells analogous to radial glial neural progenitors.

“Differential gene expression, especially in cortical radial glia, has been hypothesized to be a critical effector of brain size and a likely target of unique aspects of human brain evolution,” they write.

The authors identify several potential avenues for future investigation, such as structural variants that alter the human versions of the genes ZNHIT6, GLI3, and two key cell cycle regulators, CDC25C and WEE1. The publication also offers a significant resource to the great ape research community by annotating the ape genes and identifying full length mRNA isoforms with Iso-Seq data combined with short read RNA-seq.

The ape genomes still have some holes in comparison to human due to “upgrades” to the human reference genome using BAC-based long-read sequencing to resolve difficult, biologically relevant genomic regions such as segmental duplications. Eichler has long championed this approach and in a press release that accompanies the Science publication, he says “Our goal is to generate multiple ape genomes with as high quality as the human genome. Only then will we be able to truly understand the genetic differences that make us uniquely human.”



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星期三, 2018年6月6日

New Resource for Microbiologists: Collection of 3,000 Bacteria Genomes Released

nose bacteria

Haemophilus influenzae, a sample of which was deposited to the NCTC collection by Alexander Fleming, from his own nose.

The genomes of 3,000 strains of bacteria, including some of the deadliest in the world, are now available to researchers as part of an ambitious project by the UK’s National Collection of Type Cultures (NCTC), in partnership with the Wellcome Sanger Institute and PacBio.

Plague, cholera, streptomyces, and 250 strains of E. coli, are among the reference genomes created, as well as all ‘type strains’ of the bacteria in the collection — the first strains that describe the species and are used to classify them. The genome sequences of these highly valuable strains are fundamental for developing ways to identify specific infections in people, including tests diagnosing bacterial infections in the field to rapidly identify the source of an outbreak and help contain infections.

The collection includes several of the most important known drug-resistant bacteria, such as tuberculosis (one of the top ten causes of death worldwide, infecting 10.4 million and killing 1.7 million people in 2016 alone) and gonorrhoea (the sexually transmitted disease that infects 78 million people a year and is now becoming extremely difficult to treat) — and some varieties of historical significance, such as a dysentery-causing Shigella flexneri isolated in 1915 from a soldier in the trenches of World War 1, and a sample from the nose of penicillin discoverer Alexander Fleming.

“Historical collections such at the NCTC are of enormous value in understanding current pathogens,” said Julian Parkhill from the Wellcome Sanger Institute. “Knowing very accurately what bacteria looked like before and during the introduction of antibiotics and vaccines, and comparing them to current strains from the same collection, shows us how they have responded to these treatments. This in turn helps us develop new antibiotics and vaccines.”

“PacBio’s comprehensive DNA sequencing enables deep genomic analyses, and we are happy to be partnering with them for this important project,” he added.

Our CSO Jonas Korlach, stated: “The high-quality genomic maps enabled by SMRT Sequencing allow an unprecedented understanding of these bacteria. We are delighted to be chosen by institutions like Wellcome Sanger to help create such essential resources for the scientific and public health communities.”

Going forward, all the bacterial species in the NCTC collection will be sequenced as they are collected. Researchers can order bacterial strains from the NCTC website. Full information about each strain, including the DNA sequences, are available at EMBL-EBI.

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星期四, 2018年5月24日

Full-Length HIV Sequences Reveal Distinction Between Viruses in Brain, Other Tissue

Full-length sequencing of HIV envelope

Photo by C. Goldsmith

Scientists have made important inroads in understanding why patients with HIV develop neurological disorders despite treatments that otherwise hold the virus at bay. The project was made possible with SMRT Sequencing, which generates reads long enough to span the full HIV envelope.

Ultradeep single-molecule real-time sequencing of HIV envelope reveals complete compartmentalization of highly macrophage-tropic R5 proviral variants in brain and CXCR4-using variants in immune and peripheral tissues” was recently published in the Journal of NeuroVirology by lead author Robin Brese, senior author Susanna Lamers, and collaborators at the University of Massachusetts Medical School and Bioinfoexperts. In the article, the team describes a novel approach for examining how HIV evolves in the brain, segregated from HIV replicating in peripheral tissues. This may explain why the virus continues to attack the brain even when viral load in the rest of the body is controlled with antiretroviral therapy.

Analyzing individual virus genomes has been the preferred method for studying this phenomenon, but doing so with short-read sequencers “is problematic with HIV because millions of short sequences are generated, which subsequently require assembly, a near impossible feat with HIV [envelope] due to its high sequence variability combined with the error rate of NGS,” the scientists report. For this study, the researchers used SMRT Sequencing to generate full-length sequences of the HIV envelope, eliminating the need for assembly. Tissue samples came from a deceased, 43-year-old male HIV patient who had been responding well to drug therapy but had been diagnosed with HIV-associated dementia. Samples were collected from brain, lymph node, lung, and colon.

Scientists generated full-length envelope sequences — spanning about 2.6 kb each — and aligned nearly 53,000 unique reads. They developed phylogenetic trees showing “that brain-derived viruses were compartmentalized from virus in tissues outside the brain with high branch support,” the team writes. They further add that “variants from all peripheral tissues were intermixed on the tree but independent of the brain clades.” Finally, they note that the depth of sequencing and variation found within the brain samples was compelling, and that “SMRT did not simply reamplify thousands of sequences that were derived from a single or very few proviruses, but likely reflects the true diversity in the tissue.” Interestingly, CXCR4-using variants were found only outside the brain, while viruses within the brain used the CCR5 co-receptor.

“The study is the first to use a SMRT sequencing approach to study HIV compartmentalization in tissues and supports other reports of limited trafficking between brain and non-brain sequences during [combined antiretroviral therapy],” the scientists conclude. “Due to the long sequence length, we could observe changes along the entire envelope gene, likely caused by differential selective pressure in the brain that may contribute to neurological disease.”

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星期三, 2018年5月16日

Disease-Causing LINE-1 Insertions: Rare or Just Hard to Spot?

SMRT Sequencing has made it possible for Portuguese scientists to discover a notoriously difficult to find LINE-1 insertion mutation which caused a patient's muscular dystrophy.

Patient’s muscle biopsy – Hematoxylin and eosin stain shows severe fibrosis and fat substitution. Arrow indicates a necrotic fiber.

LINE-1 (long interspersed nuclear element) insertions cover almost 17% of the human genome, but they are notoriously difficult to resolve accurately with short-read sequencing technology, according to scientists in Portugal. That matters because intronic LINE-1 elements can cause disease. In a recent study, SMRT Sequencing made it possible to analyze the multi-kilobase region and find a mutation causing muscular dystrophy.

In “Exonization of an Intronic LINE-1 Element Causing Becker Muscular Dystrophy as a Novel Mutational Mechanism in Dystrophin Gene,” scientists from several institutes in Portugal report finding a LINE-1 insertion that disrupted an open reading frame in the dystrophin gene. Lead authors Ana Gonçalves and Jorge Oliveira, senior author Rosário Santos, and collaborators describe this work in the journal Genes.

The 50-year-old male patient suffered onset of Duchenne/Becker muscular dystrophy at age 13. Earlier attempts to identify the causative mutation — including multiplex-ligation probe amplification and genomic sequencing — had failed. The scientists used several technologies for this case, deploying SMRT Sequencing to genotype the LINE-1 element that was detected as an interruption in an open reading frame in the dystrophin gene. “An aberrant transcript was identified, containing a 103-nucleotide insertion between exons 51 and 52, with no similarity with the DMD gene,” the authors report. “This corresponded to the partial exonization of a long interspersed nuclear element.” SMRT Sequencing analysis confirmed that a full LINE-1 sequence was present, and perfectly matched an element located in chromosome 2 that might have been its source. Based on the discovery, the patient’s children were also analyzed and his daughter was found to be a carrier of the same mutation.

LINE-1 insertions within a gene are believed to be rare, with just 30 events reported in the literature, the scientists note. Most of those are found in exonic regions. Intronic LINE-1 insertions have been determined to cause disease in three cases, featuring chronic granulomatous disease, familial retinoblastoma, and Chanarin-Dorfman syndrome. It is possible that the small number of events reported is a result of technology limitations: “In the case of intronic LINE-1 insertions, detection may be hampered by the intron’s length and the fact that it mainly affects transcriptional events,” the scientists write.

“To our knowledge, this is the first report of a deep-intronic insertion of a LINE-1 element in the DMD gene shown to cause disease,” the scientists conclude. “Besides its scientific relevance … this finding also reinforces the need to develop comprehensive approaches to identify LINE-1 insertion profiles in the human genome.”

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星期一, 2018年5月14日

Codfish Study Employs Target Capture and SMRT Sequencing to Explore Evolution

Cod fishMany investigators rely on targeted sequencing approaches for deep dives into genomic regions of interest. By designing specific probes — often using short-read sequences directed towards the exome and supported by existing reference genomes or transcriptome assemblies — scientists can home in on exactly the area they want to explore.

But what about sequences in intergenic regions not covered by short reads, which could contain crucial regulatory elements varying between populations that might be of functional and evolutionary importance? Or, what about species lacking high-quality reference genomes to guide probe design?

A team of Norwegian researchers are tackling these challenges using PacBio long-read sequencing technology for their target capture experiments. In a pre-print posted on bioRxiv, corresponding author Sissel Jentoft, first author Siv Nam Khang Hoff, and colleagues at the University of Oslo, Roche NimbleGen, and Roche Diagnostics, describe how they used the technique to elucidate the evolution of the hemoglobin gene clusters in codfishes.

Hemoglobins (Hbs), key respiratory proteins in most vertebrates, are of great importance for ecological adaptation in fishes, as environmental factors such as temperature directly influence the solubility of O2 in surrounding waters and the ability of Hb to bind O2 at respiratory surfaces.

Previous studies have suggested remarkably high Hb gene copy number variation between codfish species. One study, for example, reported a negative correlation between the number of Hb genes and depth at which the species occur was observed, suggesting that the more variable environment in sunlit waters has facilitated a larger and more diverse Hb gene repertoire.

Interested in resolving the organization of Hb genes and their flanking genes in a selection of codfishes inhabiting different environmental conditions, the Oslo team turned to SMRT Sequencing to generate long, highly accurate, and continuous assemblies of these specific genomic regions of interest.

“Comparative genetic studies of gene organization or synteny requires longer, more continuous stretches of DNA containing more than one gene,” the authors explain.

Eight codfish species were selected on the basis of phylogenetic and habitat divergence. A highly continuous genome assembly of Atlantic cod (previously created using PacBio sequencing), as well as low-coverage draft genome assemblies of all eight species were used to design probes spanning both exons and introns of the genomic regions of interest. To enable targeted sequence capture for PacBio sequencing, the team used a modified protocol for sequence capture offered by Roche NimbleGen (the SeqCap EZ protocol) and generated custom barcodes.

“The generation of highly continuous assemblies enabled reconstruction of micro-synteny revealing lineage-specific gene duplications and identification of a relatively large and inter-species variable indel located in the promoter region between the Hbb1 and Hba1 genes,” the authors write.

The results shed light on the evolutionary history of Hb genes across species separated by up to 70 million years of evolution, and reveal genetic variations possibly linked to thermal adaptation, they conclude.

“Our study demonstrates that this approach… is a highly efficient and versatile method to investigate specific genomic regions of interest across distantly related species where genome sequences are lacking,” they add.

For pointers on how you can use SeqCap EZ for target sequence capture on PacBio Systems, check out this protocol.

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星期四, 2018年5月3日

Introduction of the Iso-Seq Method: State of the Art for Full-Length Transcriptome Sequencing


Photo by John Cobb on Unsplash

In eukaryotic organisms, the majority of genes are alternatively spliced to produce multiple transcript isoforms. Gene regulation through alternative splicing can dramatically increase the protein-coding potential of a genome. Therefore, understanding the functional biology of a genome requires knowing the full complement of isoforms. Microarrays and high-throughput cDNA sequencing are useful tools for studying transcriptomes, yet these technologies provide only small snippets of transcripts. Accurately reconstructing complete transcripts to study gene isoforms has been challenging [1, 2].

The Iso-Seq method produces full-length transcripts using Single Molecule, Real-Time (SMRT) Sequencing [3]. Long read lengths enable sequencing of full-length transcripts up to 10 kb or longer, eliminating the need for transcript assembly or inferencing. The Iso-Seq bioinformatics pipeline, which is freely available through SMRT Analysis, further processes the data into high-quality consensus transcript sequences that enable accurate isoform annotation and open reading frame prediction [4].

Since it does not require a reference genome or existing annotation, the Iso-Seq method has been widely adopted by the scientific community to analyze a variety of important agricultural crops and animals such as coffee, cotton, maize, rabbit, chicken, and many others. In all cases, the researchers discovered a much more diverse and complex transcriptome than previously understood. For example, Kuo et al. expanded the chicken annotation to ~64,000 transcripts, of which ~21,000 were novel lncRNAs not annotated in Ensembl. In another case, Wang et al. were able to expand and correct the maize B73 genome annotation, including the discovery of 867 novel lncRNA transcripts.

The ability to unambiguously determine the full exonic structure of complex genes, with no assembly required, also makes the Iso-Seq method attractive to the study of human diseases. Kohli et al. were able to characterize androgen receptor (AR) isoforms in castration-resistant prostate cancer to show that one novel isoform, AR-V9, was co-expressed with AR-V7 and predictive of drug resistance. Tseng et al. discovered novel splice patterns in the FMR1 gene in premutation carriers for Fragile X-associated Tremor/Ataxia syndrome that were undetected in the control group.

Perhaps somewhat surprisingly, after the Iso-Seq dataset for the MCF-7 breast cancer cell line was released to the public [5], it was revealed that this well-studied sample contained more cancer fusion genes, two new mitochondrial lncRNAs and novel sample-specific transcripts. In a recently published study, Anvar et al. used this same deep MCF-7 dataset to show that there is widespread coupling of transcript features, where more than 7,000 genes were found to have preferential coupling of 5’ start sites, exons, and polyadenylation sites. Such a study would not have been possible without the ability to precisely determine the starts and ends, as well as the splice junctions, of each transcript isoform.

But the Iso-Seq method is not just limited to eukaryotes. Recently, a new protocol called SMRT-Cappable-seq was developed to sequence the E. coli transcriptome. The result is a dramatic increase in the number of annotated operons and readthrough for the bacterium. Similarly, the Iso-Seq method was used to discover new coding and anti-sense transcripts in the previously poorly annotated human cytomegalovirus.

Since the launch of the Iso-Seq protocol in SMRT Analysis in 2014, the analysis pipeline has seen several improvements. The new Iso-Seq2 protocol, released in SMRT Analysis 5.1 last month, improves both speed and transcript recovery [6]. More importantly, over the past 5 years the bioinformatics community has embraced the technology, sparking the development of additional tools. IsoCon, IDP, and IDP-denovo are error correction methods that work for targeted genes or hybrid data. Specialized long read aligners such as minimap2 now support alternative splicing. Cupcake and TAMA are two lightweight alignment processing tool suites. SQANTI categorizes Iso-Seq transcripts against an existing annotation and combines it with short read expression data. A growing list of community tools is maintained at the Iso-Seq wiki.

We encourage our users to continue finding new ways to utilize full-length transcript sequencing with PacBio and contribute to exciting biological discoveries!


Select Publications:

  1. Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts. GigaScience 1–13 (2017). doi:10.1093/gigascience/gix086
  2. Wang, M. et al. A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation. New Phytol 217, 163–178 (2017).
  3. Wang, B. et al. Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. Nat Comms 7, 11708 (2016).
  4. Chen, S.-Y., Deng, F., Jia, X., Li, C. & Lai, S.-J. A transcriptome atlas of rabbit revealed by PacBio single-molecule long-read sequencing. Sci. Rep. 7, 1–10 (2017).
  5. Kuo, R. I. et al. Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human. BMC Genomics 18, 1–19 (2017).
  6. Kohli, M. Androgen Receptor Variant AR-V9 Is Coexpressed with AR-V7 in Prostate Cancer Metastases and Predicts Abiraterone Resistance. Clin Cancer Res 23, 1–13 (2017).
  7. Tseng, E., Tang, H.-T., AlOlaby, R. R., Hickey, L. & Tassone, F. Altered expression of the FMR1 splicing variants landscape in premutation carriers. BBA – Gene Regulatory Mechanisms 1860, 1117–1126 (2017).
  8. Weirather, J. L. et al. Characterization of fusion genes and the significantly expressed fusion isoforms in breast cancer by hybrid sequencing. Nucleic Acids Research 43, e116–e116 (2015).
  9. Gao, S. et al. Two novel lncRNAs discovered in human mitochondrial DNA using PacBio full-length transcriptome data. Mitochondrion 38, 41–47 (2018).
  10. Chakraborty, S. MCF-7 breast cancer cell line PacBio generated transcriptome has ~300 novel transcribed regions, un-annotated in both RefSeq and GENCODE, and absent in the liver, heart and brain transcriptomes. 1–8 (2017). doi:10.1101/100974
  11. Anvar, S. Y. et al. Full-length mRNA sequencing uncovers a widespread coupling between transcription initiation and mRNA processing. Genome Biol. 19, 1–18 (2018).
  12. Yan, B., Boitano, M., Clark, T. & Ettwiller, L. SMRT-Cappable-seq reveals complex operon variants in bacteria. bioRxiv 1–34 (2018). doi:10.1101/262964
  13. Balazs, Z. et al. Long-Read Sequencing of Human Cytomegalovirus Transcriptome Reveals RNA Isoforms Carrying Distinct Coding Potentials. Sci. Rep. 1–9 (2017). doi:10.1038/s41598-017-16262-z


References and Resources:

[1] Steijger, T. et al. Assessment of transcript reconstruction methods for RNA-seq. Nat Meth 10, 1177–1184 (2013).

[2] Angelini, C., Canditiis, D. & Feis, I. Computational approaches for isoform detection and estimation: good and bad news. BMC Bioinformatics 15, 135–43 (2014).

[3] Iso-Seq Template Preparation for Sequel Systems

[4] Gordon, S. P. et al. Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing. PLoS ONE 10, e0132628 (2015).

[5] PacBio MCF-7 blogpost: https://www.pacb.com/blog/data-release-human-mcf-7-transcriptome/

[6] PacBio Iso-Seq GitHub: https://github.com/PacificBiosciences/IsoSeq_SA3nUP/

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星期一, 2018年4月30日

An Interview with Baylor’s Fritz Sedlazeck on New Long-Read Algorithms

structural variation with Sniffles and NGMLR

Fritz Sedlazeck

Nature Methods just published “Accurate detection of complex structural variations using single-molecule sequencing,” a publication that presents the NGMLR aligner and Sniffles structural variant caller, both designed for use with long-read sequencing data. We chatted with developer and lead author Fritz Sedlazeck from the Human Genome Sequencing Center at Baylor to learn more.

Q: Why was a new alignment tool needed when many scientists already use BWA and other methods?

A: When I started my postdoc in Mike Schatz’s lab at Cold Spring Harbor, I had the opportunity to look at the complex SK-BR-3 cell lines. We soon discovered two challenges not addressed effectively by existing aligners: mapping split reads correctly, and handling the random short insertion and deletion errors that are characteristic of long reads.

Q: Why was Sniffles needed for structural variant detection?

A: Most of the methods for structural variant detection focus on paired-end reads. There were no appropriate structural variant calling tools at the time for long-read data, and very few callers that take into account split-read alignments. You have to have a method that parses through the full read.

Q: When you applied these tools to long-read data, what could you see that wasn’t visible before?

A: Before we started to think about how we could improve the alignments and structural variant calling, we spent a lot of time looking at IGV, focusing on single reads in complex regions like oncogenes. We knew there were some events that were hidden from us, and we saw a lot of noise coming out. That really motivated us to develop these new tools to find the signal in the noise. When we first applied them, very quickly we were detecting these structural variants. Some of the first results from Sniffles were identifications of amplification events and inversions that had not been found before.

Q: You’ve talked about plans to sequence 100 people with SMRT Sequencing from PacBio. What are the goals of that study?

A: This study is aiming at the concept of comprehensive genomes, or what Richard Gibbs calls “super-genomes.” We have SNP calls from Illumina, PacBio reads to call structural variants, and for a few samples we have 10x Genomics data for really long phasing. Our best example so far is a 67 Mb phasing block N50 for SNV and SVs. This pilot study covers many different ethnicities. The majority of samples are from African Americans, and there are many samples from Hispanic individuals as well. There are just a few Caucasians. We hope to get a good ethnicity-specific structural variant call set that we can use to inform other studies as well. We are confident that we’ll be able to identify many more structural variants that are invisible to short-read data.

Q: How much long-read coverage is needed for accurate structural variant discovery in a human genome?

A: We are aiming for about 10-fold coverage, which leaves us with 5-fold per haplotype. That’s enough for good coverage of each chromosome and lets us see the vast majority of structural variants.

For more technical detail about Sniffles and NGMLR, check out our blog post covering this paper as a preprint or attend the upcoming LabRoots webinar on May 9, in which Sedlazeck will give a talk entitled “Size Matters: Accurate Detection and Phasing of Structural Variations.”

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