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

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Thursday, July 28, 2016

The Birth of a Symbiont: Epigenome Analysis Reveals Shifting Methylation Profile

A recent publication from scientists at the University of Florida and the University of Missouri used SMRT Sequencing to analyze epigenomic changes that occur when free-living bacteria associate with a host and become symbiotic instead.

Published in the Frontiers in Microbiology journal, “Integrating DNA Methylation and Gene Expression Data in the Development of the Soybean-Bradyrhizobium N2-Fixing Symbiosis” comes from a team of collaborators including lead author Austin Davis-Richardson and senior author Eric Triplett. The scientists aimed to assess the role of epigenetics in bacterial evolution from free-living to symbiont and chose SMRT Sequencing because it generates base-specific modification information as it sequences DNA.

In its symbiotic state, the nitrogen-fixing Bradyrhizobium diazoefficiens colonizes the roots of soybean plants. Previous studies had reported significant differences in gene expression as the bacterium underwent the transition from free-living to symbiont, leading this team to investigate how much of that could be explained by changes in methylation. They sequenced a free-living bacterium and an endosymbiont, generating a single finished 9 Mb contig representing the genome of the free-living organism. This was used as a reference genome for assembly of the symbiotic strain and made it possible for scientists to identify the 681 kb symbiosis island, which contains genes known to be involved in the infection process.

The methylation analysis found 3,276 changes in five DNA motifs between the strains; 768 of them were associated with differentiation from free-living to symbiotic state, representing more than 9% of all genes and more than 35% of genes with differential expression between the strains. Of the altered genes, 80 were located in the symbiosis island but currently have no known function in the differentiation process. Of the five DNA motifs found, four had increased methylation in the free-living bacterium, and intriguingly, the fifth — the 5’-CCTTGm6AG-3’ motif — was only seen in the symbiont.

“These associations between methylation and expression changes in many B. diazoefficiens genes suggest an important role of the epigenome in bacterial differentiation to the symbiotic state,” the scientists report, noting that follow-up studies with more replicates will be needed to fully test the hypothesis.

Read More »

Tuesday, July 26, 2016

Mitochondrial Genome Analysis Yields Novel Findings in Antiretroviral-Treated HIV Patients

Scientists from Yale University and Memorial Sloan Kettering Cancer Center used SMRT Sequencing to determine whether antiretroviral therapies were triggering mitochondrial genome mutations in HIV patients. The results were recently published in HIV Medicine (“High frequency of mitochondrial DNA mutations in HIV-infected treatment-experienced individuals”).

The publication, from lead author Min Li, senior author Elijah Paintsil, and collaborators, reports results from an analysis of 71 people, including 47 HIV patients who had received antiretroviral therapy (about half had mitochondrial toxicity) and 24 healthy controls. DNA was isolated from peripheral blood mononuclear cells and mitochondrial genome sequencing performed on a PacBio System.

SMRT Sequencing was chosen for its long reads and accuracy, according to the authors. “PacBio technology overcomes some of the limitations of current next-generation sequencing platforms by providing significantly longer reads (> 1 kb), single molecule sequencing, and a single-pass error rate of < 15%,” Li et al. write. “Moreover, SMRT sequencing exceeds the consensus accuracy achieved by other sequencing methods because of the random nature of the errors. The SMRT sequencing achieves results with > 99.999% accuracy.”

The team found that HIV-infected patients, regardless of whether they experienced mitochondrial toxicity or not, had a higher frequency of mitochondrial mutations — in particular, large-scale deletions — than their healthy counterparts. However, they did not find statistically significant differences in mutation load between HIV patients with and without mitochondrial toxicity. “We did not observe mtDNA depletion in HIV-infected treatment-experienced patients with toxicity as expected,” the scientists report. “To our surprise, HIV-infected treatment-experienced patients with toxicity had significantly higher mRNA expression of Pol-γ in comparison with HIV-infected treatment-experienced patients without toxicity (P < 0.05) and HIV-uninfected controls (P < 0.01). This contradicts the Pol-γ inhibition theory.”

The authors attribute some of these novel findings to sequencing and analyzing the whole mitochondrial genome, whereas previous studies have taken less comprehensive approaches. The work also paves the way for increased use of peripheral blood mononuclear cells to characterize the biology of HIV patients, compared to the more commonly used invasive tissue biopsies.

Additional studies will be needed to follow up on these discoveries, the scientists note. “There are currently two proposed mechanisms to explain the association between mtDNA mutations and [antiretroviral therapy (ART)],” they write, “ART directly or indirectly provides a permissive environment for sporadic mtDNA mutagenesis; and ART pressure leads to clonal expansion of existing mutations.”

Read More »

Monday, July 11, 2016

First Comprehensive View of Alternative Splicing in Sorghum Powered by SMRT Sequencing

A paper from scientists at Colorado State University and the National Center for Genome Resources provides an in-depth view of the transcriptome of sorghum, a crop that’s important for human and animal food and also shows potential as a biofuel. Through this project, the team produced a new isoform analysis pipeline for community use and identified novel genes, as well as far more alternative splicing than had been expected for this plant.

The publication, “A survey of the sorghum transcriptome using single-molecule long reads,” comes from lead author Salah Abdel-Ghany, senior author Anireddy Reddy, and collaborators. The researchers were particularly interested in alternative splicing and alternative polyadenylation, two mechanisms that increase transcript diversity and may help plants adapt to stress. “Despite the fact that several large-scale RNA-seq studies have been performed in plants to analyse [alternative splicing], currently it is not known how many distinct splice isoforms are produced,” the team writes. “This is primarily due to challenges associated with short-read sequencing in accurately reconstructing full-length splice variants.”

To directly observe full-length splice isoforms, they turned to long-read SMRT Sequencing to characterize the transcriptome of sorghum seedlings. The scientists also developed the Transcriptome Analysis Pipeline for Isoform Sequencing, or TAPIS, to identify alternative splicing events and evidence of alternative polyadenylation. “The analysis of sorghum Iso-Seq data uncovered over 7,000 novel [alternative splicing] events, ~11,000 novel splice isoforms, over 2,100 novel genes and several thousand transcripts that differ in 3′ untranslated regions due to [alternative polyadenylation],” the team reports, noting that many of the novel genes are putative long non-coding transcripts. The total number of unique transcripts was nearly 28,000, covering more than 14,000 genes.

The scientists discovered a significantly higher rate of alternative splicing than had been expected for sorghum. “Previously, it was reported that pre-mRNAs of ~1,500 genes undergo [alternative splicing] in sorghum,” they note. “In this work, we demonstrate that this number is much higher.” Indeed, they found more than 10,000 alternative splicing events, compared with fewer than 3,000 included in existing gene models. The scientists performed a number of validation studies to confirm these and other novel findings and have made their TAPIS tool available for use in other organisms.

“Pacific Biosciences single-molecule long reads obtained using the Iso-Seq protocol offer a considerable advantage in transcriptome-wide identification of full-length splice isoforms and other forms of post-transcriptional regulatory events such as [alternative polyadenylation],” the team writes.

Read More »

Wednesday, July 6, 2016

In Chinese Genome Assembly, SMRT Sequencing Finds Novel Genes and Recovers Missing Sequence

ncommA paper just out in Nature Communications reports the de novo genome assembly and transcriptome of a Chinese individual, generated from long-read SMRT Sequencing and other technologies. The effort revealed nearly 13 Mb of sequence not included in the GRCh38 reference genome as well as novel gene and alternative splicing content not annotated in GENCODE.

Long-read sequencing and de novo assembly of a Chinese genome” comes from lead author Lingling Shi at Jinan University and senior author Kai Wang from the University of Southern California, as well as many other collaborators in China and the US. The team was particularly interested in finding population-specific variants, including structural variants, which required the use of long-read sequencing. Assemblies based on short-read sequence data “may have inherent technical limitations in characterizing repeat elements that span longer than the read length, yet repeats and segmental duplications are known to cover approximately half of the human genome,” the scientists write. Using SMRT Sequencing and mapping technology from BioNano Genomics, “we perform detailed characterization of the HX1 genome and demonstrate that long-read sequencing can detect functional elements in human genomes that are missed by short-read sequencing.”

For the genome assembly, the team sequenced DNA from an anonymous Chinese individual (HX1) to 103x coverage, producing a 2.93 Gb genome with a contig N50 of 8.3 Mb. Included in the results were 206 Mb of alternative haplotypes that “were constructed along with the primary contigs,” Shi et al. write. Consensus accuracy for the assembly was 99.73%, matching the accuracy of the well-known NA12878 genome assembly. In an analysis of structural variants, the team found about 20,000 insertions and deletions, with half of them classified as short tandem repeats or mobile elements. Nearly 50 exonic deletions or insertions were specific to the HX1 genome, including one previously characterized deletion that has only been seen in the Asian population.

The team also developed a new gap-filling method to make use of all this sequence data. They determined that nearly 30% of gaps in the GRCh38 reference genome could be addressed with data from HX1. “The total length of filled or shortened gaps amounts to 7.1 Mb,” they report. “We further evaluated the repeat contents within the gaps that can be closed by us, and found that simple repeats and satellite sequences were significantly enriched within the closed gaps compared with GRCh38.”

Using the Iso-Seq method, the scientists also analyzed the transcriptome of this individual and detected more than 58,000 isoforms, including “57 isoforms at 42 loci that do not overlap with any GENCODE transcript,” they write. Follow-up studies for some of the more complex data — such as “a novel transcribed element with at least five exons and six isoforms” — validated these predicted splicing events. They also found at least two genes that have never been identified with short-read data. The team looked for disease-causing variants, finding two that were classified in ClinVar as pathogenic. However, “manual review of the literature cited in the two ClinVar records indicated that both of them represented erroneous database records,” the scientists report. “This analysis highlights the need for extreme caution in interpreting ‘pathogenic’ variants documented in variant databases.”

“In summary, while short-read-based alignment and variant calling based on reference genome remain a common practice to assay personal genomes, de novo assembly by long-read sequencing may reveal novel and complementary biological insights,” Shi et al. conclude. “Furthermore, long-read RNA sequencing may identify novel transcripts that can be missed by short-read RNA sequencing.”

Read More »

Friday, June 24, 2016

Iso-Seq Study Reveals More Complexity than Expected in Maize Transcriptome

maizeIn a new publication from Cold Spring Harbor Laboratory, scientists produced a dataset for what authors call “the single largest collection of [full-length] cDNAs available in maize” and significantly improved genome annotation. The effort relied on the Iso-Seq method with SMRT Sequencing, which allows scientists to generate ultra-long reads covering full transcripts.

The paper, “Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing,” comes from lead author Bo Wang and senior author Doreen Ware, who is also affiliated with the USDA Agricultural Research Service. It offers the first published results from using the Iso-Seq method on a maize plant and includes a number of advances, such as barcoding to permit cost-effective pooling of tissue samples.

The scientists embarked on the project to see what advantages long-read sequencing offers for transcriptome analysis in a complex plant. “Although data from short-read sequencing have accumulated over recent years, they do not provide full-length (FL) sequence for each RNA, limiting their utility for defining alternatively spliced forms,” Wang et al. write. “In some cases, short-read sequencing generates low-quality transcripts, leading to incorrect annotations.” Long-read sequencing, on the other hand, makes it possible to capture high-quality, full-length transcripts.

The team used PacBio sequencing for six tissue types from the maize line B73, size-selecting libraries with the SageELF system to increase average read length. Results were impressive, with more than 110,000 transcript isoform sequences associated with nearly 27,000 genes, representing 70% of previously annotated maize genes as well as novel isoforms and even some novel genes. Long noncoding RNAs were another highlight: they found nearly 900 novel lncRNAs, many of them significantly longer than previously identified lncRNAs. “Our analysis indicates that the new transcriptome data have enormous potential to improve the current maize annotation,” the authors write. “The 111,151 unique transcripts characterized here almost double the number of transcripts documented in the RefGen_v3 annotation.”

They also analyzed alternative splicing, finding more than twice as many isoforms per gene than exist in the previous maize annotation and contributing thousands of novel isoforms to the public resource. To learn more, they studied methylation patterns associated with alternative splicing and discovered that CHG methylation appears to suppress splicing while CG methylation apparently increases the rate of splicing.

In addition to demonstrating that SMRT Sequencing data could correct mis-annotated gene models, the team showed that long reads are even more important than expected for transcriptome studies. The average transcript length in this project — almost 3 kb — is much longer than that from the previous maize annotation. “These findings show that the prevalence of long transcripts, from both coding and non-coding genes, is higher than previously thought,” Wang et al. write. “Just as the availability of short-read technologies over the last decade heralded an era of tremendous gains in small RNA research, it is reasonable to expect that long-read technologies will prompt a new focus on heretofore poorly understood characteristics of exceptionally long RNAs.”

For more, check out this case study [PDF] of Doreen Ware and the maize project.

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Thursday, June 23, 2016

Data Release: Zika-Susceptible Aedes aegypti De Novo Genome Assembly

We are excited about the release of a new genome assembly for the mosquito Aedes aegypti, which we hope will aid scientists in studying vector-pathogen dynamics, including those of the rapidly spreading Zika virus. The Aedes aegypti Aag2 cell line genome sequence was generated by a joint effort between Raul Andino’s lab at The University of California, San Francisco and PacBio. This cell line was derived by Peleg in 1975 and adapted in 1991 by Lan and Fallon. It was selected for sequencing based on its susceptibility to infection by many arboviruses, including Dengue, Chikungunya, Zika, Sindbis, and Rift Valley Fever Virus, and for its use in molecular biology and virology experimentation. Genome sequencing was performed on genomic DNA purified from cells acquired from the Gamarnik lab at the Fundación Instituto Leloir. The Aag2 cell line is also persistently infected with Cell Fusing Agent Virus, an insect-specific Flavivirus.

The de novo assembly from ~58x shotgun SMRT Sequencing long-read coverage was performed using PacBio’s FALCON assembler, followed by polishing using the Quiver consensus caller. The assembly resulted in 3752 contigs, totaling 1.72 Gigabases, with a contig N50 of 1.42 Mb. The genome size of this cell line is significantly larger than that of the Liverpool assembly, likely due to the acquisition of a mini-chromosome.

The assembly has been publicly released through VectorBase. It is our hope that this new genome assembly will be of value to the scientific community in their efforts to understand the underlying mechanisms of arboviral infection and transmission in this critical disease-carrying vector.

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Wednesday, June 22, 2016

East Coast UGM: SMRT Sequencing Data for Carnivorous Plants, Hummingbirds, Mammalian Methylation, Repeat Expansions, and More

Many thanks to the nearly 200 scientists who signed up for our East Coast User Group Meeting, and to the Institute for Genome Sciences for hosting us! The event was a hit with customers and PacBio staff alike, and discussions we heard in the hallways and during breaks told us there was some great knowledge exchange that should give labs inspiration for generating and analyzing their SMRT Sequencing data.

The day kicked off with a talk from our own Marty Badgett, senior product manager for the PacBio RS II System and Sequel System. He offered some historical perspective on throughput improvements over time for the SMRT Sequencing platforms (believe it or not, throughput has increased 100x in the past five years) and shared some data produced by the Sequel System. He spoke about the newly released SMRT Link software, which brings existing modules into an integrated workflow and adds components for data management. Badgett also alerted users to four upcoming enhancements: spin column size selection, on-chip additive cleanup, asymmetric SMRTbell templates, and active loading. With these and other changes, Badgett predicted that a 3 Gb genome could be assembled de novo with the Sequel System for $9,000 by next year and as little as $1,500 in 2018.

The event focused on our customers and the impressive results they’ve achieved with SMRT Sequencing. Jerry Jenkins from HudsonAlpha talked about high-quality de novo plant genomes — his team has released six FALCON-assembled plant genomes already, with more coming soon. The simple sequences and repetitive elements common to plant genomes are a major challenge, he said, noting that “it gives Illumina a fit and with PacBio it works well.” He said SMRT Sequencing has been “a game-changer” for his team when it comes to contiguity and completeness of assemblies. He gave several examples, including the bean Phaseolus vulgaris, for which a Sanger/454 assembly had a contig N50 of 39.5 kb while PacBio yielded 1.9 Mb. An analysis of cotton showed that a short-read assembly was missing nearly 500 Mb of sequence, which was recovered in the PacBio assembly.

Another plant presentation came from Victor Albert of the University at Buffalo, who intrigued attendees with his description of the carnivorous aquatic plant Utricularia gibba. Its genome was originally published in 2013 sporting 82 Mb in a fragmented assembly, but a new PacBio assembly shows the genome size is actually about 100 Mb and captures the data in fewer than 600 contigs. One challenge for this plant is its 3 Mb of rDNA repeats. “You can never assemble rDNA repeats,” Albert said of other sequencing platforms. “PacBio zips right through them.” He was delighted to find that the largest contig contained a full chromosome, and noted that the accuracy was excellent. “There’s no sense in doing any polishing with Illumina,” he added. With the new assembly, he’s able to explore telomeres, centromeres, and retrotransposons; he can also separate tandem duplication events from artifacts of previous whole genome duplications.

Rachael Workman from Johns Hopkins presented transcriptome data generated through the Most Interesting Genome SMRT Grant program for the ruby-throated hummingbird, Archilochus colubris. The team used the Iso-Seq method to analyze liver tissue to better understand this bird’s remarkable metabolism, finding 450,000 unique isoforms. While the full analysis is still underway, Workman shared some interesting examples, such as the complete coverage of a glucose transporter gene that shows quite a bit of divergence across avian species.

Continuing the avian theme, Duke’s Erich Jarvis spoke about the B10K project, an effort to sequence every species of bird on the planet. Jarvis’s area of interest is vocal learning, a trait shared by few organisms. His lab has found that PacBio sequencing produces genome assemblies of much higher quality than those from short-read data (in some cases increasing the contig N50 from as little as 30 kb to as much as 10 Mb, and reducing contig counts from more than 120,000 to about 1,000). In one example, he showed that a dopamine receptor that appeared to be lost by many bird species was actually conserved across them; it just wasn’t included in the short-read draft assemblies. Another example was of Egr1, with a promoter region that scientists had long wanted to study but wasn’t assembled in any existing bird genome. With PacBio sequencing, the region is fully assembled for the first time.

Tao Wu from Yale presented findings of A-base methylation in mouse embryonic stem cells (if you missed the seminal Nature paper on this previously unsuspected form of methylation in a mammal, check out our recap). Wu and team developed SMRT ChIP, a method for conducting ChIP-seq on a PacBio sequencer, and discovered N6-methyladenine throughout the genome. “Without PacBio, I think we could not get this done,” he said. The findings were confirmed by mass spec. Wu also tracked down the Alkbh1 demethylase and conducted functional studies that showed the methylation suppressed genes and transposons.

After lunch, the topic shifted to studies of primate and human genomes. Julie Karl from the University of Wisconsin analyzed the MHC locus in macaques, which have a more complex allele assortment than humans do. Using PacBio amplicon sequencing to span the full 1.1 kb locus, she was able to resolve full-length alleles unambiguously and phase haplotypes, finding many novel alleles to contribute to community databases. Karl is now working to expand her immunology investigation to KIR and FCGR, two other complex regions.

NIAID’s Brandon DeKosky spoke about sequencing antibody repertoires, which requires analyzing both heavy chain and light chain elements. Previous protocols involved analyzing three separate amplicons to cover these regions, but with the PacBio system his team has been able to sequence full-length amplicons covering both elements in B cells. “I certainly am in favor of moving everything over to the PacBio,” he said. “We can see this entire molecule at once.” DeKosky and his colleagues have studied cells from humans and rhesus macaque, and are now using transgenic mice to model antibody response to knocked-in precursor genes to determine whether this approach could be used towards developing a vaccine.

In a clinically oriented research presentation, Tetsuo Ashizawa from the Houston Methodist Research Institute reported the analysis of the ATXN10 gene responsible for spinocerebellar ataxia type 10. The gene harbors a pentanucleotide repeat in intron 9, characterized by an ATTCT repeat sometimes interrupted by other five-nucleotide repeats that lead to three broad disease phenotypes. Since the full region can’t be amplified, Ashizawa used CRISPR/Cas9 for target enrichment, making a double-strand break near the region and attaching a SMRTbell adapter for PacBio sequencing . This allowed the team to assess the motifs associated with different subtypes, in one example studying brothers with different phenotypes of the disorder and finding that the difference lay in repeat content.

Maria Nattestad from Cold Spring Harbor spoke about her genome and transcriptome analysis of SK-BR-3, one of the most widely studied breast cancer cell lines with a Her2 amplification. Long-read SMRT Sequencing was able to characterize the whole genome. Nattestad’s Assemblytics tool, which she used to call variants ranging from single-base changes to large structural changes, is now available as a web app. She discussed her reconstruction of how variants worked together to amplify the Her2 oncogene (an effort we profiled in-depth). Using the Iso-Seq method to produce full-length transcripts, she was also able to detect and explain complex gene fusions for which there was previously no DNA evidence.

Our CSO Jonas Korlach wrapped up the day with a talk highlighting some of the biggest advances we’ve seen from SMRT Sequencing, noting that there are now nearly 1,500 papers describing the use and value of this technology. Particularly productive topics include hospital-associated infections, base modifications in bacteria, and phylogenetic profiling of microbial communities. He also spoke about the recent wave of de novo human assemblies from PacBio data, noting that a shift toward diploid assemblies will be important as the community deepens its understanding of human genetic variation. Korlach also cautioned attendees about confusing contigs with scaffolds; while some assemblies boast impressive scaffold N50s, they may be missing a lot of important information if contigs are short and disconnected.

We’d like to thank our partners, who helped us put on this great event: Advanced Analytical Technologies, Covaris, Diagenode, DNAnexus, PerkinElmer, and Sage Science. With their support, we were able to host the full-day user group meeting, but also half-day workshops on sample prep and bioinformatics as well.

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Wednesday, June 15, 2016

SMRTest Microbe Grant Program:
Nominate Your Favorite Microbe Now!

We’re pleased to launch the latest opportunity to have your favorite microbe sequenced on a PacBio System. The SMRTest Microbe Grant Program, kicking off at this year’s American Society for Microbiology annual meeting in Boston, will give one scientist a free genome sequence for his or her chosen microbe. For those folks unable to make it to the meeting this year, don’t worry — you do not need to attend ASM Microbe to enter the program.

To enter, simply submit a short application describing your microbe or microbial community and how it would benefit from the long read lengths, high accuracy, and direct detection of epigenetic modifications generated by SMRT Sequencing. Entries are due by July 15, 2016.

One winner will be selected by a panel of scientists and will receive up to four library preparations and a sequencing run using up to eight SMRT Cells. Sequencing and bioinformatics analysis will be performed by our SMRT Grant co-sponsor, the Institute for Genome Sciences, a PacBio Certified Service Provider.

If you’ll be attending ASM, learn more about the SMRT Grant program by visiting the booths for PacBio (#304) or the Institute for Genome Sciences (#407). Good luck to all grant applicants!

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Friday, June 10, 2016

Case Study: Institute for Genome Sciences Expands Long-Read Capabilities with Sequel System

An updated case study about the Genomics Resource Center (GRC) at the University of Maryland’s Institute for Genome Sciences (IGS) reports that SMRT Sequencing has become an integral tool for generating complete microbial genomes, improving plant and animal genome assemblies, and exploring human genome variation.

The GRC has a scientific pedigree and a sample-to-interpretation service commitment that place it in a league of its own. The team operates under a simple mantra: ‘If it can be sequenced, we can do it.’

Both the GRC and IGS were founded in 2007 when a high-powered team of investigators formerly at The Institute for Genomic Research (TIGR), led by Claire Fraser, joined the University of Maryland School of Medicine. “The team of faculty and staff that came here to start the institute was heavily focused on infectious disease research,” says Luke Tallon, scientific director and founding leader of the GRC. “Our primary goal in joining a medical school was to extend our pathogen genomics expertise into host-pathogen studies and direct clinical genomics applications.”

In addition to its infectious disease and genomics expertise, TIGR was also renowned for its bioinformatics talent — a trait that continues with the group at the GRC. Their team of 15 staff members is evenly split between wet lab and bioinformatics, and more than half of the institute’s 100-plus employees are bioinformaticians.

The GRC was formed both to serve the genomics institute and as a university core. “We serve investigators throughout the University of Maryland system as well as across the country and around the world,” says Lisa Sadzewicz, administrative director of the facility. “Our strength is not just our deep history and experience in sequencing and genomics, but our end-to-end service level from the initial project consultation through to publication, including all of the informatics.”

Over the past five years, the GRC has applied these strengths to the PacBio platform. As early adopters of the technology, they have dedicated significant resources to the development of both laboratory and data analysis processes to leverage SMRT Sequencing. Since its adoption of the original PacBio RS in 2011, the GRC has steadily increased its utilization of the platform. In the past year alone, they have constructed more than 400 libraries and sequenced more than 1,200 SMRT Cells. These have spanned projects ranging from whole genome sequencing to metagenomics, Iso-Seq transcriptomes, and custom amplicons.

The GRC’s newest PacBio sequencer arrived in February 2016, making them one of the first PacBio Certified Service Providers to take delivery of a Sequel System. “Given our history of early adoption and success with the PacBio RS, and the promise of increased and scalable throughput, we were excited to be among the first centers to acquire a Sequel instrument,” says Dr. Fraser. The Sequel System represents the newest generation of SMRT Sequencing, providing more scalability and lower sequencing project costs compared to the PacBio RS II.

Development of processes and applications for the Sequel System is well underway at the GRC. The team plans to use the increased throughput to expand their services for Iso-Seq transcriptome sequencing and amplicon projects. In collaboration with Dr. Jacques Ravel, associate director of IGS, they are also developing a full-length 16S sequencing pipeline to complement and expand their human microbiome and metagenomic research portfolios. As read lengths and throughput on the Sequel instrument improve, Tallon’s team will shift whole genome sequencing projects onto the platform.

For other teams considering whether SMRT Sequencing is the right choice for them, Tallon says: “If you value complete genome sequences, de novo transcript discovery, and are looking at epigenetics in addition to the genome sequence, there’s no better technology out there.”

Check out the full case study to learn more about the GRC’s strength in infectious disease, development of the FDA ARGOS database, and work on large genomes.

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Tuesday, May 31, 2016

AACR Recap: Cancer Transcriptomes and a Moonshot Initiative

The PacBio team headed to New Orleans this past April to take in all the exciting new research presented at the American Association for Cancer Research Annual Meeting, show off our new Sequel instrument, and of course enjoy some crawfish and beignets!

On the first day of the conference, we had the pleasure of hearing a talk from last year’s AACR “What Will You Discover About Cancer?” SMRT Grant winners, Malgorzata Komor and Remond Fijneman from the Netherlands Cancer Institute. Malgorzata discussed her work to identify novel biomarkers to identify precursor lesions in colorectal cancer, which can be integrated into the fecal immunochemical test (FIT) used by the Dutch national health system. While the current FIT has useful sensitivity for cancer, it is only 27% sensitive for precancerous lesions. In order to uncover new biomarkers, they are mining mass spectrometry data from patient samples. However, ~50% of the spectra cannot be matched back to a known protein. In order to augment standard protein databases with peptides that may be present in precancerous cells, they have undertaken transcriptome sequencing of cell lines with disrupted splicing machinery. While they used both short-read and PacBio sequencing, they found that the Iso-Seq method particularly excelled at uncovering skipped and retained exon events. A number of the mis-splicing events found from RNA sequencing were confirmed to be translated by mass spectra. They plan to further investigate their biomarker candidates by screening additional patient samples.

Later in the session, we heard from Maria Nattestad, who discussed her work analyzing the genome and transcriptome of the breast cancer cell line SK-BR3 with PacBio long-read sequencing. In addition to presenting a compelling assembly and overview of the whole transcriptome results, she delved into the details of the extensive rearrangements of the Her2 gene in this highly aneuploid metastatic cell line. She described the use of two new software tools, Sniffles and SplitThreader, to confirm rearrangement events by split-read analysis and to visualize and explore the connectivity of the cancer genome and understand the natural history of complex structural variants.

We also saw a range of posters featuring PacBio long-read sequencing throughout the week, including posters on long-read sequencing from FFPE samples, the methylation profiles of Heliobacter pylori strains linked to gastric cancer, B-cell receptor signaling in non-GCB DLBCL, and targeted capture of 6 kb fragments for cost-effective detection and haplotype resolution of somatic cancer variants.

Finally, in a much-anticipated address at the close of the conference, Vice President Joe Biden shared his vision for a cancer moonshot with AACR attendees. He pledged to “make a decade’s worth of progress in five years” and shared ideas for accelerating the pace of research that had coalesced over the past year spent speaking to a wide range of scientists and leaders in the cancer research community. Biden has picked up the call for a number of reformist ideas that have been gaining traction in recent years within scientific community: incentivizing the sharing of data, getting publicly funded results out from behind pay walls, simplifying the grant application process, and funding reproducibility studies. With a goal of allocating $800 million in new funding for his initiative, including $600 million for the National Cancer Institute, Biden’s ideas for removing some of the inefficiencies of our science infrastructure should have some heft behind them. He also highlighted a number of research areas as priorities for new funding, including enhanced and early detection technology, cancer vaccine development, cancer immunotherapy and combination therapy, genomic analysis of tumor and surrounding cells, and pediatric cancer research.

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Thursday, May 26, 2016

Save the Spuds: UK Team Identifies Blight-Resistance Genes with SMRT Sequencing

Potato with late blight disease

Potato with late blight disease

Scientists from the UK published new work detailing important advances in protecting potatoes from the disease that caused the Irish potato famine in the 1800s. It’s not just of historical interest; the team points out that late blight disease is once again endangering the food supply, with global yields of potatoes shrinking in recent years.

Accelerated cloning of a potato late blight–resistance gene using RenSeq and SMRT sequencing,” published in Nature Biotechnology, comes from lead authors Kamil Witek and Florian Jupe, senior author Jonathan Jones, and collaborators at The Sainsbury Laboratory and The Genome Analysis Centre.

In the paper, the scientists describe an ongoing search among wild potato species for genetic resistance to Phytophthora infestans, the pathogen responsible for late blight disease. For this work, they looked for NLR genes (encoding nucleotide-bind­ing, leucine-rich repeat proteins that are essential for activating plant defense mechanisms) in Solanum americanum, a wild Mexican potato relative. The process of enriching for resistance genes, which they call RenSeq, originally began with short-read sequencing but encountered difficulties in de novo assembly of NLR genes due to highly repetitive sequence and gene copies.

“To accelerate [resistance] gene cloning, and to remove any need for construction of a bacterial artificial chromosome or fosmid libraries, we refined RenSeq to capture and sequence fragments of up to 3.2 kb, which is the average NLR gene length,” the authors report. They incorporated SMRT Sequencing and found that most molecules were covered multiple times in individual reads, leading to highly accurate results.

In a comparison of short-read and long-read NLR assemblies, the scientists found that the short-read versions were as fragmented as they suspected, with just 21% of contigs including a full-length NLR gene. They determined that more than 300 long-read sequences were not represented in short-read data and identified several chimeric sequences in the short-read contigs. “The central positions of matching, aligned CLC or SPAdes NLR assemblies to long-read contigs further confirmed the superiority of SMRT RenSeq by also capturing >1 kb of flanking promoter and terminator sequences,” Witek et al. write. “Capture and sequencing of long fragments can resolve any repetitive gene family or structural genome variation by spanning repeat-rich regions with long reads.”

The team says this approach could be very useful in general for rapid engineering of pathogen-resistant crops. “The SMRT RenSeq method has the potential for use in investigating genetic variation for other important traits likely to involve known multigene families such as metabolic pathways (e.g., cytochromes P450, terpene cyclases) or transcription factors, especially if combined with mutagenesis,” they conclude.

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Wednesday, May 18, 2016

Discover New Insights into Human Genetic Variation at ESHG in Barcelona

The PacBio team is looking forward to joining 3,000 other scientists in Barcelona May 21-24 for the European Human Genetics Conference, better known as ESHG. Organized by the European Society of Human Genetics, this is the 49th year of a high-quality meeting where the latest developments in human and medical genetics are discussed.

This year, we’ll be showcasing our new Sequel System at booth #260 in the exhibit hall. Come visit us and learn more about it! With higher throughput than our previous instrument, we think the Sequel System will be a great fit for the genomics community on projects such as multiplex targeted sequencing and RNA isoform sequencing.

To learn how scientists are already using PacBio sequencing to address unanswered questions in genomics, don’t miss the SMRT Sequencing workshop hosted by Roche Sequencing. The luncheon event will be held on Monday, May 23, from 11:15 a.m. to 12:45 p.m. in rooms 120 and 121. Christine Beck from Baylor College of Medicine will discuss the use of long fragment capture and sequencing techniques to reveal structural variation at clinically relevant loci. Robert Sebra from the Icahn School of Medicine at Mount Sinai will discuss how his lab uses long-read sequencing to gain a more comprehensive view of complex regions of the genome, including pharmalogically important sites, oncogenes, and structural variants linked to genetic disease.

There will also be ESHG talks and posters featuring SMRT Sequencing data in a wide range of applications, including whole genome assembly and haplotyping, immunology, repeat expansion disorders, and non-coding RNAs. Please join us at the following presentations:

 

Saturday, May 21, 2016, 10:30 a.m. – 12:00 p.m.

Talk Title: E01.1 Long-read sequencing of complex genomes

Speaker: Evan Eichler, University of Washington

 

Saturday, May 21, 2016, 6:30 p.m. – 8:00 p.m.

Talk Title: A distinct class of chromoanagenesis events characterized by focal copy number gains

Speaker: Matthew Hestand, Leuven, Belgium

 

Sunday, May 22, 2016, 1:00 p.m. – 2:30 p.m.

Talk Title: Enrichment of unamplified DNA and long-read SMRT Sequencing to unlock repeat expansion disorders

Speaker: Tyson Clark, Pacific Biosciences

 

Sunday, May 22, 2016, 1:00 p.m. – 2:30 p.m.

Talk Title: C07.6 – Detection of AGG interruptions in FMR1 premutation females by single-molecule sequencing

Speaker: S. Ardui, KU Leuven

 

Monday, May 23, 2016, 8:30 a.m. – 10:00 a.m.

Talk Title: S11.3 – Mapping Human Long Noncoding RNAs

Speaker: Rory Johnson, Barcelona, Spain

 

Tuesday, May 24, 2016, 11:00 a.m. – 12:30 p.m.

Talk Title: C23.6 – Identifying novel long non-coding RNAs in the Human genome.

Speaker: M. P. Hardy, Wellcome Trust Sanger Institute

 

POSTERS

 

Saturday, May 21, 12:00 p.m. – 2:00 p.m.

Monday, May 23, 10:15 a.m. – 11:15 a.m.

Presentation: P16.07C – Application Specific Barcoding Strategies for SMRT Sequencing

 

Saturday, May 21, 12:00 p.m. – 2:00 p.m.

Sunday, May 22, 4:45 p.m. – 5:45 p.m.

Presentation: P16.02B – Highly Contiguous de novo Human Genome Assembly and Long-Range

Haplotype Phasing Using SMRT Sequencing

 

Sunday, May 22, 10:15 a.m. – 11:15 a.m.

Presentation: P07.17A – Resolving KIR genotypes and haplotypes simultaneously using Single-

Molecule, Real-Time Sequencing

 

Sunday, May 22, 4:45 p.m. – 5:45 p.m.

Presentation: P15.14B – Full-length and phased CYP2D6 variant genotyping using the PacBio RS II

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Tuesday, May 17, 2016

Arizona Scientists Deploy BAC Expertise and SMRT Sequencing for Crop Genomes

At the University of Arizona, a leading genomics research facility benefits from decades of BAC-based sequencing expertise, original studies of crop genomes, and a unique emphasis on high molecular weight DNA. Rod Wing, founding director of the Arizona Genomics Institute (AGI) and a professor in the School of Plant Sciences, Ecology & Evolutionary Biology at the university, was a pioneer in building BAC-based reference genomes in the ’90s. Today, that carefully honed expertise in isolating large DNA fragments gives him and his lab a real advantage for making the most of long-read sequencing.

Wing’s efforts primarily focus on plant genomes, but his service facility performs genomic studies on a wide variety of organisms for investigators at the university and around the world. He says the PacBio platform is useful for customers interested in sequencing any kind of crop, as well as animals and other organisms. “If your genome is littered with repetitive elements that are highly similar, PacBio allows you to get through those elements and back into some more unique sequence for a better assembly,” he notes. “Our facility can work on pretty much any organism — we just have to have some good DNA.”

To meet his own research goal of building high-quality reference genomes for every species of rice, 23 in total, Wing chose SMRT Sequencing. “PacBio is revolutionizing our approach for whole genome shotgun, BAC, and targeted sequencing,” he says. The rice genome assemblies his team is building will be essential to improving rice crops for higher yield, expanded growing areas, and stress resistance.

In recent work to sequence the African rice genome, a few highly repetitive and rearranged BACs were particularly challenging to get through with other platforms. “It was taking us months to try to get through this one region,” Wing recalls. Using SMRT Sequencing, he produced the full sequence of this region “in a nice single piece within a couple of days.” For particularly difficult regions like that one, PacBio sequencing is a very powerful targeted approach, he adds. With a few other rice genomes, his team is pooling BACs, 32 at a time, in individual SMRT Cells. “Around 85 to 90 percent of those BACs are completely circularized,” he says. “It’s pretty easy to go through a genome using this approach.”

Beyond the highly accurate, long-read sequence data he obtains from SMRT Sequencing, Wing also likes the PacBio platform for full-length isoform sequencing and its ability to characterize the methylome. He notes that now they can take rice tissues at several developmental stages and under many different environmental conditions, isolate RNA, and do Iso-Seq analysis on those samples to enable whole plant transcriptome analysis. This could help the community map gene networks and pinpoint the biological mechanisms behind traits such as bigger leaves, water uptake, and more.

To learn more about Wing’s rice genome projects and his service facility, check out this case study.

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Thursday, May 12, 2016

From Seabass to Salmon:
Swimming in High-Quality Genomes

CC BY 2.5, https://commons.wikimedia.org/w/index.php?curid=1654077

Asian seabass

A global collaboration of researchers has produced what is likely the most contiguous assembly of a fish genome to date. “Chromosomal-Level Assembly of the Asian Seabass Genome Using Long Sequence Reads and Multi-layered Scaffolding,” published in PLoS Genetics, comes from lead author Shubha Vij and senior author László Orbán with collaborators at nearly two dozen labs.

The team set out to sequence Lates calcarifer, the Asian seabass, which has a genome of about 670 Mb grouped into 24 A chromosomes and as many as 10 B chromosomes. They used SMRT Sequencing from PacBio to overcome the fragmented and incomplete assemblies associated with short-read data, and incorporated optical and genetic mapping to add additional layers of information to the assembly. At the end, they generated an incredibly valuable resource that has been shared with the community. “The quality of the Asian seabass genome assembly far exceeds that of any other fish species, and will serve as a new standard for fish genomics,” the authors write.

That quality largely came from 90x SMRT Sequencing coverage of the genome, which on its own produced an assembly with about 3,900 contigs and a contig N50 length greater than 1 Mb. Layering in optical mapping and genetic markers allowed the team to place contigs into larger scaffolds, ultimately achieving 24 individual chromosomal scaffolds. The scientists anticipate this resource will be particularly useful for comparative genomics and for development of assays such as GWAS, allele mining, and genomic selection.

The scientists used the MHC region for a close-up inspection of the genome quality and contiguity. They compared the seabass MHC locus to that of stickleback, which had “the most complete published fish genome assembly available in the public repositories” at the time of the study, the team reports. In Asian seabass, they identified 14 MHC class I genes, spread across eight contigs, half of which were longer than 1 Mb. “By contrast, the MHC-class I genes from stickleback, were located on almost double the number of contigs, of which all except one were ≤ 113 kb in length,” they write.

The remarkable assembly quality gave researchers the opportunity to delve into repeat sequences, which represent nearly 20% of the genome and included several kinds of complex tandem repeat sequences. In addition, the team studied duplicated genes and found they were enriched for functions that could help explain how the fish transforms from male to female after maturity.

The scientists note that while most eukaryotic genomes published so far have been produced with short-read data, a strategy using only PacBio data “seems ideal for assembling mid-to-large eukaryotic genomes since it ensures contiguity, less ambiguity and assembly metrics surpassing all of the fish genomes sequenced thus far.”

And if you just can’t get enough fish genomes, don’t miss this publication reporting a new salmon assembly. The team used PacBio sequencing as part of a multi-platform assembly process; the findings shed light on the rediploidization of salmon.

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Wednesday, May 4, 2016

Eco-friendly Soil Remediation Gets a Boost with the Latest SMRT Grant Program Winner

Congratulations to the winner of the first-ever SMRT Grant program decided by the community: Renying Zhuo of the Chinese Academy of Forestry!

We ran polling through our Genome Galaxy Initiative on the Experiment crowdfunding platform and were amazed to see much how it galvanized the genomics community. There were 30,000+ responses to the competition across the five finalists for our “Explore Your Most Interesting Genome” grant opportunity. Zhuo garnered the most support for his project to sequence two highly related strains of Sedum alfredii for a comparative genomics investigation to identify key genes important for remediating soil contaminated by heavy metals. This project could be applied to address cadmium ion pollution, a growing concern within rapidly industrializing nations.

The remaining finalists have a second chance to earn funding by launching their projects through a special crowdfunding event. The runners-up will kick off their campaigns through Experiment, and we sincerely hope that supporters of these worthy projects come back to help. Every donation makes a difference! And, donations are only accepted when projects meet their crowdfunding goal. Here’s a quick look at the projects now open for contributions:

The Amazing & Enigmatic Alpaca

Investigator: Kylie Munyard, Curtin University

According to Munyard’s proposal, the economically important alpacas are of great scientific interest on a number of fronts, and producing a reference genome will enable new studies in both agricultural and biomedical research. Alpacas are a good model for diabetes research; they have innate mechanisms to stay free of parasites; their distant relationship to other agricultural animals makes them good for comparative study; and much more.

Sequencing an Extremophile Earthworm

Investigator: Luis Cunha, Cardiff University

This project would sequence the extraordinary earthworm Pontoscolex corethrurus, which lives in a volcanic geothermal field with high exposure to toxic gases, extreme temperatures, and very little oxygen. Cunha’s proposal notes that preliminary work with draft assemblies indicates significant levels of horizontal gene transfer that could be better characterized with SMRT Sequencing.

Scar-Free Regeneration in the Spiny Mouse

Investigator: William Barbazuk, University of Florida

According to this proposal, the adult spiny mouse is the only known mammal with the unique ability to regenerate skin and organs after wounds without any scars or other indications of trauma, making this organism interesting for regenerative medicine. Barbazuk hypothesizes novel genes, alternatively-spliced isoforms, and gene expression regulators are responsible. He aims to use SMRT Sequencing to study the transcriptome of spiny mouse and its wound-healing properties.

Highlighting Firefly: A Genome Resource

Investigator: Jing-Ke Weng, MIT

This project would help a large consortium of researchers generate a high-quality genome assembly for Photinus pyralis, an American firefly. Weng’s proposal notes that the 2,000-plus species of these charismatic flashing beetles have been understudied, and that the biological mechanisms behind important traits such as bioluminescence remain unknown.

The PacBio team is very grateful to the scientific community and their supporters, as well as our co-sponsors for this grant program: Sage Science, Computomics, RTL Genomics, Texas A&M AgriLife Genomics and Bioinformatics Service, and Experiment.

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Monday, May 2, 2016

Join the SMRT Community: User Meetings in Europe, Asia, and the US

There are several PacBio user meetings coming up, and with locations around the world we hope you’ll be able to attend one of them. These meetings are a great way to meet fellow customers, exchange tips, and learn about new applications. If you are interested, please register as soon as possible to reserve your seat.

 

The Netherlands: SMRT Leiden Symposium & Informatics Developers Meeting, June 6-8

This meeting, organized and hosted by Leiden University Medical Center’s human genetics department, includes a scientific symposium and the first SMRT Informatics Developers meeting in Europe. There’s an impressive agenda for the two-day symposium, with topics covering genomics, transcriptomics, and epigenomics of organisms ranging from microbes to humans. Keynote speakers include Evan Eichler, Steven Marsh, Shinichi Morishita, and Hagen Tilgner. On June 8, the informatics conference will kick off with a keynote talk from Gene Myers and provide plenty of opportunity for brainstorming about approaches to de novo assembly, structural variant detection, genome phasing, the Iso-Seq method, and more. Registration is free.

 

Maryland: East Coast User Group Meeting & Workshops, June 7-9

PacBio’s fourth annual East Coast User Group Meeting will be hosted by the University of Maryland’s Institute for Genome Sciences. In addition to the day-long meeting on June 8, attendees may also participate in half-day workshops on sample prep (June 7) and bioinformatics (June 9). The sample prep workshop will cover best practices and basic data analysis, with group breakout discussions for deeper dives into specific topics of interest. The bioinformatics event will introduce SMRT Analysis 3.x, including the SMRT Link GUI and command line examples. Data management for both the PacBio RS II and the new Sequel System will be discussed. Reserve your seat now.

 

Singapore: Asia User Group Meeting & Workshop, June 8-10

This event, held at the Grand Copthorne Waterfront Hotel, will feature two days of presentations for the user group meeting and an additional day-long bioinformatics workshop. The general meeting will include topics from de novo genome assembly and targeted sequencing to Iso-Seq full-length RNA sequencing and epigenomics, covering microbes, plants, animals, and humans. The bioinformatics workshop will focus on genome assembly and quality control, targeted sequencing and phasing, detection of minor variants or structural variants, and more. Learn more and sign up for the meeting.

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Wednesday, April 27, 2016

Upcoming Webinars on Biomedical Research, Data Analysis, and Structural Variation

We’ve got several educational webinars coming up, and we hope you can join us!

Our first event will be hosted by Front Line Genomics on April 28 (4:00 p.m. BST / 11:00 a.m. EST / 8:00 a.m. PST). “Applying PacBio Long-Read Sequencing for Human Biomedical Research” will include Adam Ameur of the National Genomics Infrastructure in Sweden; Giancarlo Russo from the Functional Genomics Center Zurich; and our CSO Jonas Korlach. Each participant will offer a brief presentation, with audience Q&A at the end.

dnanexus logoWe’ve also teamed up with DNAnexus to offer two webinars on best practices for SMRT Sequencing data analysis. The first, on May 4 (5:00 p.m. CET / 11:00 a.m. EST / 8:00 a.m. PST), features DNAnexus Computational Biology Project Leader Brett Hannigan discussing rapid assembly for reference-quality genomes. The webinar will include a look at the challenges involved in assembling the 4.5 Gb tobacco genome and walk through running the FALCON assembler on the DNAnexus platform.

The second webinar, focused on discovering structural variants in SMRT Sequencing data, will take place on June 16 (5:00 p.m. CET / 11:00 a.m. EST / 8:00 a.m. PST). Andrew Carroll, Director of Science at DNAnexus, will talk about using cloud-optimized apps such as PBHoney, Parliament, and Sniffles to improve the accuracy of calling structural variation.

All webinars will also be recorded. If you cannot attend in person, please sign up and we will send you the file following the event.

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Monday, April 25, 2016

On DNA Day Honoring Discoveries – Y chromosome, Reference Grade De Novo Assemblies & Methylation

98074_thumbHappy DNA Day, everyone! This scientific celebration has us reflecting on the many advancements the community has made in the past year. For a molecule that is sequenced thousands of times a day all over the world, there is still much to learn. Today we’d like to honor some of the remarkable science enabled by SMRT Sequencing since last year’s DNA Day.

 

Scientists have continued to make progress exploring regions of the genome that have long been considered intractable. Two of our favorite stories this year came from the always-challenging Y chromosome. Researchers studying the mosquitoes that carry malaria — Anopheles gambiae — delivered the first detailed analysis of their Y chromosome, which is essentially a giant string of repeat sequences. The information may prove essential for efforts to shift the sex ratio of mosquito populations toward males, which do not transmit disease. In a separate study, scientists analyzed the Y chromosome in Drosophila and found evidence of an ancient gene duplication from an autosome; the gene had since acquired a new function on the Y chromosome. The gene had never been discovered before because of its location in a highly repetitive, complex genomic region that was inaccessible to other sequencers.

 

We’ve also seen a number of great examples of reference-grade de novo genome assemblies in the past year. A large team of scientists produced what they called “the most contiguous clone-free human genome assembly to date” using SMRT Sequencing along with single-molecule genome maps from BioNano Genomics. A similar strategy was used to generate “a gapless telomere-to-telomere genome assembly” of the filamentous fungus Verticillium dahliae, according to the publication in mBio. Just recently researchers published a new assembly for the gorilla genome, representing better than 150-fold improvement over the previous assembly. We loved the story of Oropetium thomaeum, a resurrection grass that was sequenced for one of our SMRT Grant winners and resulted in a virtually complete assembly.

 

DNA methylation has been another area of interesting developments as scientists delve into this poorly understood genetic mechanism. A recent Joint Genome Institute project involved a sweeping analysis of 230 prokaryotes that revealed more methylation, and more complex patterns, than ever suspected. A separate study detected methylation for the first time in C. elegans, proving that even well-characterized organisms still have secrets to reveal. Scientists also made progress in understanding the role of epigenetics in virulence and antibiotic resistance; this study found an epigenetic switch in non-typeable Haemophilus influenza that alters the organism’s pathogenicity and drug resistance.

 

The past year has also been an exciting time for the PacBio team. In October, we launched our new Sequel System, a sequencer one-third the size and half the cost of the PacBio RS II with nearly seven-fold higher throughput. And right now we’re gathering votes for our first-ever community poll to award a new SMRT Grant. If you haven’t voted yet, now’s the time!

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Wednesday, April 20, 2016

Benchmarking Study:
Full-Length 16S Sequencing Offers Better Phylogenetic Resolution

Scientists from the Joint Genome Institute and other institutions recently reported a new SMRT Sequencing approach to microbial profiling using full-length sequencing of the 16S rRNA gene. In a benchmarking study, they demonstrate that this method allows for more accurate taxonomic classification than is possible with typical short-read sequencing methods.

Lead author Esther Singer, senior author Tanja Woyke, and collaborators at USDA-ARS, the University of British Columbia, and other research groups published “High-resolution phylogenetic microbial community profiling” in The ISME Journal earlier this year. The scientists note that while 16S phylogenetic analysis has traditionally been performed with gold-quality Sanger sequencing, the need for a more cost-effective solution drove the field to short-read sequencing technologies, which have produced most of the 16S sequences in GenBank. However, that shift came at the cost of quality. “Reference sequences with low read accuracy, chimeric sequences and partial rRNA gene sequences with reduced phylogenetic resolution generated on short-read sequencing platforms such as 454 and Illumina remain problematic, resulting in incorrect or less accurate classification of environmental sequences,” the authors report.

The team thought long reads from SMRT Sequencing could provide an appealing alternative. In this project, they generated full-length 16S sequences from microbial communities using a PacBio instrument and compared results to those from a short-read platform. They first tested the approach on a mock community of 26 bacterial and archaeal species including E. coli and strains of Salmonella and Clostridium, generating full-length 16S sequences called PhyloTags in a successful validation of the method.

Next they went to the field, using PacBio and short-read sequencing to analyze microbial communities from a lake in British Columbia, with water samples taken at eight different depths. They determined that partial sequences from the 16S gene — the information generated by sequencers that can’t cover the full gene in a single read — were less likely to resolve phylogeny and were more likely to lead to incorrect matches, particularly in more complex microbial communities. As many as 4% of short-read results “were taxonomically unresolved at the phylum level, whereas all PhyloTags were classified into distinct bacterial phyla,” the scientists report. In an analysis of unclustered sequence data, they note that short-read sequence results were “more often either impossible or incorrect, significantly altering community profiles across all taxonomic levels.” They also found that certain phyla were more likely to be misclassified when only partial gene coverage was available. “PhyloTag sequencing … offers the highest contig accuracy without discrimination against GC-rich or -poor regions, which further reduces bias in amplicon-based profiling,” the authors write.

“A resurgence of [full-length] sequences used as ‘gold standards’ has the potential to yet again transform microbial community studies, increasing the accuracy of taxonomic assignments for known and novel branches in the tree of life on previously unobtainable scales,” Singer et al. report.

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Monday, April 18, 2016

First Comprehensive Analysis of Mosquito Y Chromosome Offers Clues for Vector Control

pnasA new PNAS paper offers the first detailed analysis of the Anopheles gambiae Y chromosome, which could prove critical for biological and infectious disease research. The report uncovered extensive remodeling of the Y chromosome, which consists almost entirely of highly repetitive sequence. The authors say this study “provides a long-awaited foundation for studying male mosquito biology, and will inform novel mosquito control strategies based on the manipulation of Y chromosomes.”

Radical remodeling of the Y chromosome in a recent radiation of malaria mosquitoes” comes from lead authors Andrew Brantley Hall, Philippos-Aris Papathanos, Atashi Sharma, and Changde Cheng, along with senior author Nora Besansky. This large collaboration combines scientific expertise from Virginia Polytechnic Institute and State University, the University of Notre Dame, NHGRI, Indiana University, and several other institutes. The project was formed to address the lack of information about the mosquito Y chromosome, which has hindered vector-control efforts. Previous sequencing initiatives had analyzed A. gambiae but reported only 180 kb of unordered sequence data for the Y chromosome; a related mosquito genome project revealed 57 short sequences, while some 200 kb of sequence data was generated from BAC clones for the chromosome.

The challenge lies in the highly repetitive sequences found in Y chromosomes across organisms. “The Y chromosome remains one of the most recalcitrant and poorly characterized portions of any genome more than a decade into the postgenomic era,” the authors write. Among mosquitoes, the Y chromosome is particularly interesting because males do not transmit disease, so shifting the sex ratio of populations is a promising vector-control approach for reducing the incidence of malaria, Zika virus, and other mosquito-borne disease.

The team used SMRT Sequencing to tackle the A. gambiae Y chromosome, first generating a 294 Mb de novo assembly followed by sequencing and completely assembling BAC clones. “We find that the A. gambiae Y consists almost entirely of a few massively amplified, tandemly arrayed repeats, some of which can recombine with similar repeats on the X chromosome,” the scientists report.

For further analysis, the scientists incorporated genome resequencing data from a recent species radiation and determined that the Y chromosome experiences rapid sequence turnover. They also used RNA-seq data to identify a small number of genes on the chromosome that had no homologs on the X chromosome. In addition, they found YG2, a conserved gene that may have a role in sex determination in the mosquito.

The authors note that SMRT Sequencing has been a game-changing development for analyzing Y chromosomes in many organisms, “promising a resource-efficient alternative” to the laborious processes used in the past. “Single-molecule sequencing reads were able to reveal complex repeat structures from whole-genome data and completely assemble heterochromatic BACs without manual finishing,” the scientists conclude. “These results suggest that continued single-molecule read length and throughput improvements may soon enable the complete reconstruction of Y chromosomes from whole-genome data alone.”

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