This blog features voices from PacBio — and our partners and colleagues — discussing the latest research, publications, and updates about SMRT Sequencing. Check back regularly or sign up to have our blog posts delivered directly to your inbox.
Search PacBio’s Blog
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.
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.
- 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.
- 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.
- 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.
- 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.
- 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.
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.
In 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.”
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.
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.”
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.”
Many 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.
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 . 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 .
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 , 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 . 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!
- Long-read sequencing of the coffee bean transcriptome reveals the diversity of full-length transcripts. GigaScience 1–13 (2017). doi:10.1093/gigascience/gix086
- Wang, M. et al. A global survey of alternative splicing in allopolyploid cotton: landscape, complexity and regulation. New Phytol 217, 163–178 (2017).
- Wang, B. et al. Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing. Nat Comms 7, 11708 (2016).
- 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).
- Kuo, R. I. et al. Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human. BMC Genomics 18, 1–19 (2017).
- 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).
- 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).
- 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).
- Gao, S. et al. Two novel lncRNAs discovered in human mitochondrial DNA using PacBio full-length transcriptome data. Mitochondrion 38, 41–47 (2018).
- 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
- 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).
- 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
- 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:
 Steijger, T. et al. Assessment of transcript reconstruction methods for RNA-seq. Nat Meth 10, 1177–1184 (2013).
 Angelini, C., Canditiis, D. & Feis, I. Computational approaches for isoform detection and estimation: good and bad news. BMC Bioinformatics 15, 135–43 (2014).
 Gordon, S. P. et al. Widespread Polycistronic Transcripts in Fungi Revealed by Single-Molecule mRNA Sequencing. PLoS ONE 10, e0132628 (2015).
 PacBio MCF-7 blogpost: https://www.pacb.com/blog/data-release-human-mcf-7-transcriptome/
 PacBio Iso-Seq GitHub: https://github.com/PacificBiosciences/IsoSeq_SA3nUP/
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.”
The PacBio team is just back from Chicago, where we saw outstanding talks and posters at the American Association for Cancer Research (AACR) Annual Meeting and enjoyed that city’s well-deserved reputation for exciting weather. We hope everyone remembered to pack their hats and gloves and enjoyed the late-season snow!
This year multiple researchers presented work featuring the use of the Iso-Seq method for full-length transcript sequencing in cancer research. The first was a poster presented by Yeung Ho from University of Minnesota, entitled ‘The role of androgen receptor variant AR-V9 in prostate cancer’. The poster describes their discovery that the previously reported structure of AR-V9 was incorrect, and that past experiments characterizing AR-V9 expression patterns had not distinguished between it and the related isoform AR-V7. Read the full publication in Clinical Cancer Research to learn more.
Liqing Tian and Jinghui Zhang from St. Jude’s Children’s Research Hospital also presented work featuring SMRT Sequencing. In their poster on ‘Allelic specificity of immunoglobulin heavy chain (IGH) translocation in B-cell acute lymphoblastic leukemia (B-ALL) unveiled by long-read sequencing’ they shared follow up studies validating a tantalizing lead they discovered in the NALM-6 cell line transcriptome data collected in collaboration with us using the simplified Iso-Seq sample preparation protocol for the Sequel System introduced at last year’s AACR meeting. They showed that though IGH-DUX4 fusion occurs frequently and is thought to be a driver of B-ALL, DUX4 has translocated into the IGH enhancer that is repressed by immunoglobulin allelic exclusion. They theorized that translocation into the dormant enhancer may be favored either because DUX4 is toxic to the pre-B cell or because the translocation blocks VDJ recombination. Without functional B cell receptor, such clones would not survive.
Later in the day, our own Meredith Ashby presented a poster detailing recent improvements to the Iso-Seq analysis workflow, increasing both the speed and reliability of the pipeline that will benefit Sequel System users. The new pipeline can process 1 Sequel SMRT Cell in ~5 hours, and 3 SMRT Cells of data in ~13 hours. In addition, the poster described improved precision in isoform detection with the use of synthetic spike-in variants. Meredith also shared new whole transcriptome data from the breast cancer cell line HCC-1954 and normal breast tissue control, demonstrating that the Iso-Seq method reveals a surprising abundance of both novel isoforms and novel junctions.
On Tuesday afternoon, Neetu Singh from King George’s Medical University presented her poster ‘Characterization of oral squamous cell carcinoma transcriptome through long read sequencing technology’. Neetu reported numerous novel isoforms discovered in patient samples using the Iso-Seq method, including transcripts of genes previously implicated in tumor development. For example, she found that tumor samples but not controls expressed novel isoforms of SCAMP3 and type I keratin KRT-17. Similarly, she found many isoforms of KRT6, KRT14, and KRT16 gene fusions.
Interested in the Iso-Seq method for your project? We invite you to enter our upcoming Iso-Seq SMRT Grant Program: Connect the Dots with the Iso-Seq Method. As highlighted in the terrific science presented by PacBio users at AACR this year, full-length transcript sequencing with PacBio allows users to see the complete picture of splice variants, novel isoforms, and gene fusions. Tell us how the Iso-Seq method for RNA sequencing will drive new discoveries in your human-focused research for a chance to win sequencing on the Sequel System. Visit the grant submission website to learn more and get your entry ready for when the SMRT Grant opens May 1.
Structural variants account for most of the base pairs that differ between human genomes, and are known to cause more than 1,000 genetic disorders, including ALS, schizophrenia, and hereditary cancer. Yet they remain overlooked in human genetic research studies due to inherent challenges of short-read sequencing methods to resolve complex variants, which often involve repetitive DNA.
At a recent webinar co-hosted by Nature Research, Professor Alexander Hoischen joined Principal Scientist Aaron Wenger to discuss how advances in long-read sequencing and structural variant calling algorithms have made it possible to affordably detect the more than 20,000 such variants that are now known to exist in a human genome.
Wenger described methods for calling and visualizing structural variants from low-coverage, long-read sequencing of human genomes, and presented optimal study designs for both gene discovery and population genetics, while Hoischen shared case studies.
New Insights into Neurodevelopmental Disorders
Hoischen’s team at Radboud University Medical Center uses intellectual disability as a model for severe, sporadic neurodevelopmental disorders. Extensive research suggests that more than 60% of moderate to severe cases of intellectual disability are caused by de novo mutations, but even after the application of microarrays, exome sequencing, and short-read whole genome sequencing, 38% of patients remain undiagnosed because no causal variant can be found.
To address these unexplained cases, Hoischen and his colleagues adopted SMRT Sequencing to see if long-read technology could offer new insight. They already knew that information about structural variants associated with intellectual disability was lacking compared to single-nucleotide variants.
In a pilot project that is still underway, Hoischen selected five patient/parent trios and sequenced them to high coverage with the Sequel System. While all 15 people had previously been analyzed with microarrays, exome sequencing, and short-read WGS, SMRT Sequencing still uncovered 21 Mb of genomic sequence in each sample that was essentially new data. Of that, 7 Mb of sequence falls in genic space.
Using PBSV and a new joint calling tool the team beta tested, they found as many as 23,000 structural variants larger than 50 bp per genome. Nearly 70% of those variants were missed by short-read sequencing — 80% of insertions and 55% of deletions were novel, Hoischen said. By broadening their search to variants as small as 20 bp, the team expanded its variant calls to as many as 40,000 in each genome, with similar stats for novel findings. They have used PCR and other approaches to validate many of the calls, showing that the PacBio data is highly accurate.
For analysis, Hoischen and his team are focusing on de novo mutations. They use data from the parents to rapidly filter out inherited mutations, getting the patient’s universe of potentially causative variants down to very manageable numbers for follow-up study. In one example, he showed that just 40 structural variants were left to investigate in the patient after this filtering process. Hoischen said this approach is likely to be powerful for clinical applications.
Deeper Dive into Autism Spectrum Disorder
Another project was also announced during the presentation. Stephen Scherer, director of The Centre for Applied Genomics at The Hospital for Sick Children (SickKids) and Professor of Medicine at the University of Toronto, was named as recipient of the Structural Variant SMRT Grant Program, launched in partnership with GENEWIZ during the American Society of Human Genetics Annual Meeting in October 2017. He will receive sequencing on the Sequel System and bioinformatics support to pursue the project entitled “Using Low-Coverage PacBio SMRT Sequencing to Find Structural Variation Mutations in Autism Families with Multiple Affected Individuals.”
Scherer previously published results of a study in which he used short-read whole-genome sequencing to detect single nucleotide variations, indels, and copy number variations in more than 5,000 samples from families with ASD. Although he was able to successfully identify many variants and associated genes affecting autism risk, the study did not report on structural variation findings and for most families the genetic determinants are still to be resolved.
Congratulations to Dr. Scherer and we look forward to successfully applying low-coverage whole genome SMRT Sequencing to this important and ground-breaking research.
Pop quiz: Which animal accounts for around 20% of all living mammals, harbors (yet survives) some of the world’s deadliest diseases, lives proportionately longer than humans given its body size, and helps make tequila possible?
From the tiniest bumblebee bat (Craseonycteris thonglongyai) to the large (1kg) golden-capped fruitbat (Acerodon jubatus), the diversity and rare adaptations in bats have both fascinated and terrified people for centuries. Now, an international consortium of bat biologists, computational scientists, conservation organizations, and genome technologists has set out to decode the genomes of all 1,300 species of bats using SMRT Sequencing and other technologies.
The aim of the Bat1K initiative, as set forth by Emma Teeling of the University of Dublin, Sonja Vernes of the Max Planck Institute, and 146 others in this paper in the Annual Review of Animal Biosciences, is to “catalog the unique genetic diversity present in all living bats to better understand the molecular basis of their unique adaptations; uncover their evolutionary history; link genotype with phenotype; and ultimately better understand, promote, and conserve bats.”
The large sequencing project will be accomplished in three phases, starting with 21 representatives of each bat family, followed by 220 representatives for every genus of bat, and then the remaining 1,288 of the species. It will greatly expand upon the 14 bat genome assemblies currently available from the National Center for Biotechnology Information (NCBI) database, which are of varying quality and completeness.
“One primary goal of Bat1K is to standardize assembly strategies to provide assemblies of uniform optimal quality for the bat genomics community through combining multiple sequencing and scaffolding technologies,” the authors write. “We believe it is important not just to generate genome-level data, but to produce high-quality genome sequences that maximize the usefulness and accessibility of the data for all research fields.”
The bat clade exhibits a wide range of chromosomal variation. High-quality, chromosome-level genome assemblies across the group will allow researchers to investigate things like evolutionary trajectories of autosomal and sex chromosomes from nucleotide, syntenic, and phylogenomic perspectives.
The team is also hoping to resolve “some of the most passionate debates in science” centered around the evolutionary history of bats, which has been difficult to piece together due to an impoverished fossil record.
The information they uncover could benefit not only the research community, but the world at large. The authors argue that studying bats will enable us to address some of the most important challenges facing humanity into the next century including improving the well-being of a large and rapidly aging human population, preventing the spread of emergent infectious diseases, maintaining agricultural productivity, and restoring natural ecosystems worldwide.
Bats are suspected reservoirs for some of the deadliest viral diseases, including Ebola, SARS (severe acute respiratory syndrome), rabies, and MERS (Middle East respiratory syndrome coronavirus). But they appear to be asymptomatic and survive these infections. Figuring out why could increase our understanding of immune function and help prevent viral spillovers into humans.
Bats also exhibit extraordinary longevity—they can live up to 10 times longer than expected given their small body size and high metabolic rate. Only 19 mammal species are known to live proportionately longer than humans given their body size, and 18 of these are bats.
“Bats show few signs of senescence and low to negligible rates of cancer, suggesting they have also evolved unique mechanisms to extend their health spans, rendering them excellent models to study extended mammalian longevity and ageing,” the team writes.
By identifying bats’ cellular repair mechanisms, researchers could also gain insight into inflammatory disorders associated with autoimmune diseases, which are among the fastest growing causes of disease worldwide.
“The ability to modulate inappropriate inflammation in response to stressors without impairing immune function could improve the lives of millions,” the authors write.
Studying the genetics of echolocation, vocal learning, and sensory perception in bats could shed light into human blindness, deafness, and speech disorders, they add. And characterizing bat wing development could improve our understanding of how changes in limb developmental building blocks can lead to human limb malformations.
In regard to the ecosystem, bats perform key services. They pollinate crop species in the tropics (including agave, making possible the distillation of tequila) and disperse seeds across long distances, maintaining plant genetic diversity and aiding the regeneration of forests after clearing. They are able to breach ocean barriers, making them indispensable to isolated island ecosystems. They also feed on crop pests throughout their range; without bats, it is estimated that the United States would spend more than $3 billion a year on pesticides alone, the authors report.
“Bat1K will develop a genomic ark that can be used to benchmark the genomic health of different bat species to uncover populations in need of immediate conservation efforts,” the authors write. “Prioritization of bat genomes is not just desirable but indispensable to confront the many challenges to human well-being, ecosystem function, and biodiversity conservation we now face.”
Catch one of the Bat1K project leaders, Sonja Vernes, as a keynote speaker at the 2018 SMRT Leiden Conference, to be held in the Netherlands June 12-14. The meeting includes two back-to-back events: SMRT Scientific Symposium and the SMRT Informatics Developers Meeting. View the preliminary agenda and register
We’re told to avoid sugar and refined carbohydrates if we want our teeth to remain strong and cavity-free. But what is the role of microbiota in our oral health?
Cavities – or caries – actually occur as the result of bacterial infection that leads to sustained decalcification of tooth enamel and the layer beneath it, the dentin. Left unchecked, it can reach the tooth’s inner layer, with its soft pulp and sensitive nerve fibers, and, in some cases, can cause serious complications such as phylogenetic osteomyelitis and the life-threatening bacterial endocarditis.
In addition to diet and host factors, the occurrence and development of dental caries seems to be closely related to the imbalance of the oral microbiota. With this in mind, researchers at Zhejiang University in Hangzhou, China, wanted to create a profile of oral microbiota in early childhood caries, and they turned to PacBio SMRT Sequencing to do so.
As detailed in a paper published in the Frontiers in Microbiology, lead author Hui Chen, first author Yuan Wang, and colleagues derived 876 species from 13 known bacterial phyla and 110 genera from saliva samples collected from 41 Chinese preschoolers, aged 3–5 years old (21 with severe early childhood caries, and 20 who were caries-free).
A shift in the oral core microbiota was observed in the two groups, allowing the researchers to identify both protective and destructive bacteria.
“Our findings indicate that dental caries have a microbial component, which might have potential therapeutic implications,” the authors write.
At the species level, 38 species, including Streptococcus spp., Prevotella spp., and Lactobacillus spp., showed higher abundance in the caries group compared to the caries-free group. This suggests these bacteria may be risk factors for dental caries in children, the authors state.
The researchers also collected samples from the same children six months later. New cavities were developing in 5 children who were initially caries-free. Analyzing their microbiota, the researchers found that 6 species of bacteria that were abundant in the caries-free children, including Abiotrophia spp. and Neisseria spp., were much less abundant in these cases. Those bacteria were also less abundant in the initial caries group, leading the researchers to associate the strains with a healthy oral microbial ecosystem.
The authors say they chose single-molecule real-time sequencing because of its richness and resolution. Previous studies have explored the relationship between microorganisms and the development of caries; however, most of the cariogenic bacteria were only identified at the genus level, they noted.
“Species-level and even strain-level resolution is thought to be important for caries prognosis,” the authors state. “PacBio outperformed other sequencers… in terms of the length of reads, and it reconstructed the greatest portion of the 16S rRNA genome when sequencing the oral microbiota.”
At the HudsonAlpha Institute for Biotechnology, scientists are building on advances in agricultural research to power a clinical pediatric program. For this work, they’re using the Sequel System to perform whole-genome sequencing on trios of children with developmental disabilities and their parents.
HudsonAlpha researchers have been using SMRT Sequencing to resolve challenging plant genomes, deploying a Sequel System and a PacBio RS II for these complex projects. The successfulness of that program led the institute to add a second Sequel System for clinical use.
The organization is part of the NIH-funded Clinical Sequencing Exploratory Research Program, with faculty investigator Greg Cooper leading an effort to apply whole genome sequencing to better understand the genetic basis of intellectual and developmental disabilities in children and to provide diagnostic information to affected families. More than 500 children and their parents have been enrolled in the study.
In a statement announcing this work, Cooper said, “By applying whole genome PacBio Sequencing in this study we hope to more sensitively identify all sizes of genetic variants, thereby increasing our solve rate for previously undiagnosed children. In many cases, an accurate clinical diagnosis can improve our ability to manage the child’s condition. We also anticipate that we will make novel discoveries through this work that may benefit many families beyond those directly tested here.”
The group’s efforts to diagnose children using short-read sequencing technology have achieved a success rate of about 30 percent, but it is widely known that these platforms are unable to detect certain types of variation that contribute to disease. Structural variants such as repeat expansions and copy number variations are larger and more complex than short-read sequencers can resolve, and likely represent some of the cases that have gone undiagnosed. With PacBio long-read sequencing, scientists may be able to produce diagnostic answers for cases that have proven intractable with other technologies.
“We believe projects like HudsonAlpha’s CSER program to help solve undiagnosed genetic disease in children are among the most important and rewarding uses for our technology,” stated Kevin Corcoran, our Senior Vice President for Market Development. “We look forward to seeing how PacBio sequencing can both improve their diagnostic success rate as well as support new discoveries.”
Revered around the world, rice is a staple food for nearly half of the population. But as that population grows, rice breeders are faced with the challenge of producing crops that are high yielding, disease-resistant and nutritious, while at the same time being more sustainable.
The International Oryza Map Alignment Project (OMAP) was initiated in 2003 to develop a set of high-quality genomic resources for the wild relatives of rice that could be used as a resource to discover and utilize novel genes, traits and/or genomic regions for crop improvement and basic research.
Members of the consortium recently released new reference assemblies for six wild Oryza species (O. nivara, O. rufipogon, O. barthii, O. glumaepatula, O. meridionalis and O. punctata), two domesticates (O. sativa vg. indica (IR 8) and O. sativa vg. aus (N 22)) and the closely related outgroup species L. perrieri.
In a paper published in Nature Genetics, “Genomes of 13 domesticated and wild rice relatives highlight genetic conservation, turnover and innovation across the genus Oryza,” senior author Rod A. Wing, of the Arizona Genomics Institute at the University of Arizona, first author Joshua C. Stein, of Cold Spring Harbor, and colleagues from 17 other institutions, describe what they found when analyzing the new assemblies and comparing them to four previously published genomes.
Among the major findings were the identification of several disease resistance genes and haplotypes, which could support the breeding of new varieties for natural resistance to growing pathogen threats such as blast (Magnaporthe oryzae).
“Our sequencing of seven wild relatives of crop species opens a treasure trove of novel resistance haplotypes and loci to sustain this strategy,” the authors write.
“The practical utility of our resources is directly demonstrated by our identification of a strong candidate for the long-sought Pi-ta2 locus, which in combination with Pi-ta provides broad-specificity resistance to M. oryzae,” they add.
The study is also the first to contain a complete long-read assembly of IR 8 ‘Miracle Rice’, which relieved famine and drove the Green Revolution in Asia 50 years ago.
And it should prove valuable for the study of molecular evolution. As the authors note, the new dataset represents “a genome-wide vista of the results of ~15 million years of both natural and artificial selection on a single genus.”
Over this time period, the Oryzeae have maintained a base chromosome number of 12, despite their global distribution and bursts of transposable element diversification that, in some cases, led to doubling of genome sizes, the study found.
The reference genomes span the species tree, and were used to resolve several areas of the Oryza phylogeny. Their genome-based age estimates imply a “remarkably rapid diversification rate” (~0.50 net new species/million years), placing it on par with many rapidly diversifying taxa in island and continental hotspots, the authors state.
“Our phylogenomic work illustrates both the challenges of inferring species phylogenies in closely related plant taxa—incomplete lineage sorting, hybridization and introgression—and the power of whole-genome sequences to untangle the resulting phylogenetic discordance,” they write.
The amount and richness of data provided by long-read sequencing led to “a much more nuanced view… that reflects the mosaic history of different parts of the genome,” the author add.
A publication from the Molecular Plant journal demonstrates the use of SMRT Sequencing to characterize activity of transposable elements in Magnaporthe oryzae, the destructive fungus responsible for rice blast disease. This information will help scientists better understand pathogen biology and potentially find new ways to reduce its impact on an important food source.
Lead authors Jiandong Bao, Meilian Chen, Zhenhui Zhong, Wei Tang, senior author Zonghua Wang, and collaborators at Fujian Agriculture and Forestry University and Minjiang University report their findings in “PacBio Sequencing Reveals Transposable Element as a Key Contributor to Genomic Plasticity and Virulence Variation in Magnaporthe oryzae.”
They embarked on the study because “the sustainable cultivation of rice, which serves as staple food crop for more than half of the world’s population, is under serious threat due the huge yield losses inflicted by the rice blast disease,” they write. Until this project, however, some 50 previous short-read genome assemblies were not of sufficient quality to support the kinds of in-depth investigations required to understand the pathogen’s genetic mechanisms or variation across species. These assemblies “are highly fragmented and lack most of the lineage-specific (LS) regions which are more plastic than the core genome and enriched with repeats and effector proteins,” the scientists explain.
To build a better assembly, the team applied PacBio long-read sequencing to the challenge. They produced high-quality, nearly complete genome representations for two M. oryzae isolates. The resulting assemblies were far more contiguous than previous ones, with contig N50s increased to 3.28 Mb and 4.13 Mb, compared to 180 kb and 156 kb respectively for short-read assemblies. That led to a “>95% reduction in genome fragmentation,” the scientists report, and “approximately 98% of the PacBio assembled contigs were longer than 100 kb.” Alignment to the reference genome filled about 70% of sequence gaps and “confirmed that PacBio assemblies have sufficient genome coverage and superior integrity,” the team adds.
Importantly, the PacBio assemblies were about 10% larger than the short-read assemblies. Analysis of this “showed that the increased size of PacBio assembled genome was not accompanied by a corresponding increase in the number of new genes, but was as a result of significant increase in the recovery of repeat sequence,” the scientists write. That new content included many transposable elements, with some entirely novel elements detected. The scientists also analyzed the effects of transposable elements and determined that they “play a key role in regulating genomic plasticity, promote chromosome rearrangement and presence/absence polymorphism of [secreted protein] genes,” the team writes.
This study offers strong validation of the importance of transposable elements in pathogen virulence and demonstrates the utility of SMRT Sequencing for achieving high-quality assemblies to fully represent these elements.
In an exciting new Cell paper, scientists report identification of an intronic structural variant that causes a neurodegenerative Mendelian disorder that primarily affects people on the island of Panay in the Philippines. The team used a number of approaches, including SMRT Sequencing and the Iso-Seq method, to solve the medical mystery.
“Dissecting the Causal Mechanism of X-Linked Dystonia-Parkinsonism by Integrating Genome and Transcriptome Assembly” comes from lead authors Tatsiana Aneichyk, William Hendriks, Rachita Yadav, David Shin, and Dadi Gao; senior authors Cristopher Bragg and Michael Talkowski; and many collaborators at Massachusetts General Hospital, the Broad Institute, and other organizations.
The team targeted X-linked dystonia-parkinsonism (XDP), “an adult-onset neurodegenerative disease that has challenged conventional gene discovery for several decades.” Endemic to the island of Panay, the progressive disease was previously associated with several genetic variants, but none were deemed definitively causative. Scientists attribute that in part to a lack of solid annotation for this genomic region.
“We investigated XDP as an exemplar of an unsolved Mendelian disorder arising from a founder haplotype in an isolate population,” the team writes. “We hypothesized that the genetic diversity of XDP has not been captured by previous approaches and that unbiased assembly of the genome and transcriptome spanning the XDP haplotype could reveal additional sequences or aberrant transcripts unique to probands.” While most Mendelian analyses to date have used exomes or whole genome sequencing on short read platforms, the disease causing variation in the case of XDP is difficult to detect by these methods. To that end, they applied a bevy of sequencing and analysis tools — including SMRT Sequencing, hybridization capture sequencing and scaffolding technologies — to study a large cohort of about 800 individuals, most of them affected males or carriers.
“Our results identified previously unknown genomic variants and assembled transcripts that were shared among XDP probands, but not observed in controls, including aberrant splicing and partial retention of intronic sequence proximal to the disease-specific SVA [SINE-VNTR-Alu retrotransposon] insertion in TAF1,” the scientists report. SMRT Sequencing of BAC clones from a proband generated a 200 kb region spanning TAF1, assembling the full SVA sequence.
TAF1 is a general transcription factor encoded on the human X chromosome and is expressed is all tissue types, but in the case of XDP, a portion of the mRNA transcript is spliced in a non-functional manner within the intron containing the SVA. The team followed up on this observation with CRISPR/Cas9 editing to remove the SVA sequence in cell models derived from patient samples. Removal of the SVA by gene editing “rescued this XDP-specific transcriptional signature and normalized TAF1 expression,” proving that this mobile element really is the causal agent, the authors write.
“These data suggest that XDP may join a growing list of human diseases involving defective RNA splicing, [intron retention], and transcriptional alterations driven by transposable elements,” the team concludes. “These studies also illustrate the potential for layered genomic analyses to provide a roadmap for unsolved Mendelian disorders that is capable of simultaneously capturing coding and noncoding regulatory variation and interpreting their functional consequences in human disease.”
Genomic data standards will be essential for continuing the growth of genomics and ensuring its smooth transition into the clinic, according to a new Bio-IT World article written by PacBio scientist Aaron Wenger. The piece nicely sums up recent efforts from the Genome in a Bottle Consortium, the Genome Reference Consortium, and the Global Alliance for Genomics and Health to paint a picture of the state of genomic data standard development today.
“The more we learn about the human genome, the more needs we identify for data standards,” Wenger reports. “For example, early efforts focused on ensuring that single nucleotide variant (SNV) calls could be tested for accuracy; today we know that structural variants, which are responsible for the vast majority of base pair differences between any two people, are just as critical to call with precision.”
While the consortia and other collaborative programs working to fine-tune data standards are doing excellent work, Wenger notes that it is important that the initiatives avoid multiple, possibly competing, standards. “It will be essential for these large consortia to collaborate with each other to ensure that appropriate data standards are available for various needs (such as supporting both SNVs and structural variants) and that the specific use for each is clearly defined,” he writes.
He concludes with a call to all genomic scientists to get involved in these types of efforts. Data standards “will help the genomics community cross an important threshold, from the realm of pioneering tinkerers to a robust, reliable, and highly accurate science that can be readily applied both in research and in the clinic,” Wenger adds.
Genetic knowledge is powerful when it comes to breeding. The ability to trace desirable traits to the gene level can help create plants and animals that are adapted to existing and emerging challenges, such as temperature tolerance, productivity, or disease resistance.
By crossing two breeds of cattle, Angus (Bos taurus taurus) and Brahman (Bos taurus indicus), from opposite ends of the species spectrum, breeders can benefit from the Angus’s high productivity in cool environments and the Brahman’s tolerance for harsh, hot climates and the diseases and parasites found there.
Genetically and phenotypically, the two subspecies are very different. And, their offspring are as well, as John Williams of the University of Adelaide explained in his recent talk at PAG. There are even differences depending on how the breeds are crossed (i.e. Angus bull and Brahman cow, or Brahman bull and Angus cow). Fetal weight at mid-gestation, for instance, varies markedly among purebreds and crosses, and between crosses.
Interested in exploring these differences, Williams and colleagues embarked on two approaches to assembling this heterozygous genome.
The first was a one-technology methodology involving PacBio long-read sequencing, assembled with FALCON-Unzip and scaffolded with Hi-C data, to examine the genome of an F1 cross-breed (Angus x Brahman). The Iso-Seq method was then used to explore the cattle’s transcriptome. It enabled the team to examine entire transcripts, as well as isolate 30,000 isoforms from 12,000 genes.
Although they are still sifting through the data, Williams said the team is “starting to be able to differentiate between Angus and Brahman specific transcripts.”
“Initial results show that Iso-Seq data can be haplotyped and is highly concordant with genome phasing results, revealing possible allelic-specific isoform expression,” he added.
Mapped back to the assembly, the Iso-Seq data also confirmed that the F1 cattle reference genome is of good quality.
Among the genes they explored, 10 were heavily differentially expressed between male and female. The team wanted to drill down deeper, to determine which parent of origin the differences come from, and to create better assemblies of sex specific genes.
So, a sub group led by Adam Phillippy, Sergey Koren, and Arang Rhie of the National Human Genome Research Institute (NHGRI) in Bethesda, Maryland, created a new process that took advantage of access to the cattle’s parents.
Trio Binning: Two Genomes From One Individual
The “trio binning” process, also presented at PAG, enabled them to generate two high-quality (maternal and paternal) genomes from the single F1 cross-breed. It uses short reads from two parental genomes to partition SMRT Sequencing long reads from an offspring into haplotype-specific sets prior to assembly. Each haplotype is then assembled independently using a new module of the Canu assembler the NHGRI team created — TrioCanu — resulting in a complete diploid reconstruction.
As described in this preprint, the method requires moderate coverage of short sequencing reads (e.g. 30-fold Illumina) from two parental genomes to identify short, k-length subsequences (k-mers) that are specific to each parent. These k-mers are presumed to be specific to the corresponding haplotypes of the offspring. Next, long reads are collected from an offspring of the parents to sufficiently cover both haplotypes (e.g. 80-fold PacBio, 40-fold per haplotype). Long reads from the offspring are then binned into paternal and maternal groups based on the presence of the haplotype-specific k-mers and assembled separately.
In the case of the cattle, the Angus and Brahman haplotypes aligned to one another with 99.35% identity and contained 25,245 haplotype-specific structural variants and 124 inversion breakpoints.
Phillippy et al. note that trios have long been used in genomics to infer inheritance, including for the HapMap and the 1000 Genomes projects, as well as by trio-sga to simplify heterozygous diploid genome assembly. But reliance on short-read sequencing limited the haplotype-specific contigs (haplotigs) to an average size of a few kilobases.
“In contrast, our long-read method enables the assembly of multi-megabase haplotigs and complete parental haplotypes,” the authors write.
Long-read trio binning is also advantageous because it requires fewer resources than inbreeding, simplifies assembly graphs, and can accurately reconstruct structurally heterozygous alleles that can be important factors in adaptation and immunity, Phillipy states.
“Accurate representation of haplotypes is essential for studies of intraspecific variation, chromosome evolution, and allele-specific expression,” the authors add.
Its applications could also spread into human and other areas of agricultural genomics, including polyploid plant genomes.
“Reference genome projects have historically selected inbred individuals to minimize heterozygosity and simplify assembly,” the authors write. “We challenge this dogma and present a new approach designed specifically for heterozygous genomes.”
A new preprint from scientists at Uppsala University’s SciLifeLab reports the de novo genome sequencing and assembly of two Swedish individuals using PacBio SMRT Sequencing. By comparing the Swedish genomes to the human reference (GRCh38), the team found a substantial amount of novel sequence which is not present in the reference – along with over 17,000 structural variants. Further comparison of the Swedish genomes to other population-specific reference genome assemblies – including a Korean and a Chinese genome – identified novel sequences that appear to be population-specific as well as several megabases that seem to be more universal in the human genome.
“De novo assembly of two Swedish genomes reveals missing segments from the human GRCh38 reference and improves variant calling of population-scale sequencing data” comes from lead author Adam Ameur, senior author Ulf Gyllensten, and collaborators. They aimed to produce higher-quality genome assemblies than are possible with short-read sequencing technologies. “PacBio’s single-molecule real-time (SMRT) sequencing technology has proven to be an excellent method for de novo genome assembly,” they write. “The human de novo assemblies available based on long-read data … indicate that each personal genome contains a significant amount of ‘dark matter’ of structural variation that is not detected by short-read WGS.”
The scientists generated about 78-fold SMRT Sequencing coverage of each genome (one male and one female), followed by optical mapping for scaffolding. The PacBio-only assemblies were highly contiguous: authors report that each one “contained about 3,000 primary contigs and an additional 4,000 alternative contigs originating from regions with high heterozygosity.” For primary contigs, N50 values were 9.5 Mb and 8.5 Mb.
They then compared the assemblies to each other, to GRCh38, and to other de novo assemblies recently produced with SMRT Sequencing. More sequence matched between the two Swedish genomes than with GRCh38, “suggesting that the [human] reference does not contain all sequences present in these Swedish individuals,” the scientists report, citing about 10 Mb absent from the reference. Of the novel sequences discovered, about 6 Mb aligned to a Chinese genome assembly, indicating that much of the data missing from GRCh38 is not specific to the Swedish population. Novel sequences had the typical hallmarks that make detection difficult for short-read sequencers: they “are highly repetitive, have elevated GC-content and are primarily located in centromeric or telomeric regions,” according to the preprint.
“Inclusion of these novel sequences into the GRCh38 reference radically improves the alignment and variant calling of whole-genome sequencing data at several genomic loci,” the scientists add. By re-analyzing 200 samples from a short-read-based Swedish population study, the team found more than 75,000 putative novel SNVs in each person and removed 10,000 SNV calls per person that had been false positives.
“The benefits of an improved reference are likely to be even stronger for other, non-European, population groups that were poorly represented in the original assembly of GRCh38. Despite all efforts to refine the human genome since its original release in 2001, our results indicate that substantial improvements could still be made … by de novo assembly of representative human genomes from different populations,” the scientists conclude.