PacBio Blog

Wednesday, November 12, 2014

New Transcript Study Offers Clues to Pathogenesis of Repeat Disorders Linked to FMR1

It’s been nearly two years since a team of scientists from the University of California, Davis, School of Medicine published the first-ever complete sequence of FMR1, the gene associated with a repeat expansion that causes Fragile X syndrome. That team is once again breaking new ground, this time characterizing alternative splicing and full-length transcripts of FMR1. For both studies, the scientists relied on Single Molecule, Real-Time (SMRT®) Sequencing because its uniquely long reads allowed them to span the gene and generate sequence and isoform data that would not have been possible any other way.

The new paper, “Differential increases of specific FMR1 mRNA isoforms in premutation carriers,” was published in the Journal of Medical Genetics and comes from lead author Dalyir Pretto and senior author Flora Tassone, along with collaborators. They aimed to elucidate the transcript levels of FMR1 in people with what’s known as a premutation allele — people who have more repeats within the FMR1 gene than normal, but fewer repeats than full-mutation Fragile X patients have. This group is at risk for fragile X-associated tremor/ataxia syndrome as well as fragile X-associated primary ovarian insufficiency.

Earlier studies using qRT-PCR had found significant levels of alternative splicing in FMR1, but “PCR-based methodologies only allow the individual splice sites to be analysed separately and therefore fail to provide the combinations of different splice sites within the same RNA molecule,” Pretto et al. report. “Our approach, which has used the single-molecule long-read sequencing technology, has allowed us to obtain a transcript map of all of the splice combinations within a single FMR1 transcript, and more importantly in both premutation and control individuals.” Their goal was to quantify isoforms to understand their role in pathogenesis for disorders associated with premutation carriers.

The authors’ findings included some unexpected results. For example, they detected alternative splicing around exon 3 of FMR1, a region where splicing had not previously been found or even predicted. They also found that only two of the 17 exons had alternative start sites. Overall, they noted that while “the relative abundance of all mRNA isoforms was significantly increased in the premutation group,” two particular isoforms were most increased, indicating possible functional relevance for premutation disorder pathology.

As these represent the first comprehensive transcript findings for the FMR1 gene, the scientists underscore the need for future studies to more fully characterize the downstream effects of the transcripts they sequenced.

Monday, November 10, 2014

Nature Paper Offers Novel Sequence, Structural Variant Data for a More Complete Human Genome

A new paper out in Nature extends our view into the human genome and challenges current ideas about genetic variation. “Resolving the complexity of the human genome using single-molecule sequencing” comes from first author Mark Chaisson, senior author Evan Eichler, and their collaborators at the University of Washington, University of Bari Aldo Moro, and University of Pittsburgh. In the paper, the scientists describe an important effort to fill gaps and better characterize structural variation in the human genome by using Single Molecule, Real-Time (SMRT®) Sequencing data.

The team sequenced a haploid human genome, using a hydatidiform mole cell line (CHM1), to about 40x coverage. Eichler’s group was able to close or shrink 55 percent of the 160 euchromatic gaps existing in the reference genome, the vast majority of them marked by GC-rich regions with several kilobases of short tandem repeats. The approach used repeated rounds of mapping and assembling data, and added more than 1 Mb of novel sequence — including novel exons and putative regulatory sequences — to the genome.


Wednesday, October 29, 2014

‘Revolutionizing HLA Typing': Uppsala’s Ulf Gyllensten on How Long Reads Give Access to New Areas of the Human Genome

In a recent interview with Theral Timpson — part of Mendelspod’s series on long-read sequencing — Ulf Gyllensten, a scientist at Uppsala University, spoke about using PacBio® technology for HLA typing, human genome studies, transcriptomics, and more.

Based in the medical genetics and genomics department, Gyllensten focuses on two areas: using systems biology to study biological variation in human physiology and studying the epidemiology of human papilloma virus and its genetic link to cervical cancer. He also works with the National Genomics Infrastructure, a national core facility in Sweden for genotyping and DNA sequencing, where he has access to all commercially available sequencing platforms.

In the podcast, Gyllensten spoke about advances in screening for HPV, his predictions for the widespread use of genome sequencing in the clinic, and applications using Single Molecule, Real-Time (SMRT®) Sequencing for human genome studies.


Tuesday, October 21, 2014

Data Release: Whole Human Transcriptome from Brain, Heart, and Liver

In higher eukaryotic organisms, like humans, RNA transcripts from the vast majority of genes are alternatively spliced. Alternative splicing dramatically increases the protein-coding potential of eukaryotic genomes and its regulation is often specific to a given tissue or developmental stage.

Using our updated Iso-Seq™ sample preparation protocol, we have generated a dataset containing the full-length whole transcriptome from three diverse human tissues (brain, heart, and liver). The updated version of the Iso-Seq method incorporates the use of a new PCR polymerase that improves the representation of larger transcripts, enabling sequencing of cDNAs of nearly 10 kb in length. The inclusion of multiple sample types makes this dataset ideal for exploring differential alternative splicing events. Download the polished, full-length transcript sequences and the raw data files.


Monday, October 20, 2014

SMRT Sequencing for the HLA Complex: PacBio Goes to ASHI

This week marks the 40th annual meeting of the American Society for Histocompatibility and Immunogenetics, better known in the community as ASHI. The PacBio® team is looking forward to attending; after all, several organizations are now using Single Molecule, Real-Time (SMRT®) Sequencing specifically for resolving the incredibly complex genetic regions related to histocompatibility.

Earlier this year, we announced that two leading HLA typing institutions had adopted SMRT Sequencing to untangle this highly polymorphic set of genes: Anthony Nolan, a UK-based blood cancer charity that started the world’s first bone marrow registry, and HistoGenetics, a pioneer that has used sequence-based typing to characterize HLA regions in more than 14 million samples. We’re pleased to report that scientists from both organizations will be giving presentations at our ASHI workshop, Advances in Fully Phased HLA & KIR Typing Using SMRT® Sequencing.


Wednesday, October 15, 2014

New Chemistry Boosts Average Read Length to 10 kb – 15 kb for PacBio® RS II

We are pleased to announce the launch of our new reagent kit, P6-C4, which represents the next generation of our polymerase as well as our chemistry. This kit replaces the P5-C3 chemistry and is recommended for all SMRT® Sequencing applications, including de novo assembly, targeted sequencing, isoform sequencing, minor variant detection, scaffolding, long-repeat spanning, SNP phasing, and structural variant analysis.

P6-C4 continues the steady read length improvement our users have seen since the instrument first launched. With this new chemistry, average read lengths increase to 10 kb - 15 kb, with half of all data in reads 14 kb or longer. The throughput is expected to be between 500 million to 1 billion bases per SMRT Cell, depending on the sample being sequenced. By providing more throughput per instrument run, the chemistry enables users to sequence larger genomes and observe previously undetected structural variants, highly repetitive regions, and distant genetic elements.


Friday, October 10, 2014

ASHG 2014: A New Look at the Human Genome with Long-Read Sequencing

Scientists around the world are getting ready for the annual meeting of the American Society of Human Genetics taking place October 18-22 at the San Diego Convention Center. We’re looking forward to a number of excellent presentations and posters, and are delighted to see that many of them will focus on applying Single Molecule, Real-Time (SMRT®) Sequencing to human studies.

If you’ll be among those attending ASHG, be sure to attend our workshop, A New Look at the Human Genome – Novel Insights with Long-Read PacBio Sequencing, taking place 12:30 – 2:00 p.m. on Tuesday, October 21. Register in advance to reserve your seat or to receive the recording following the event. Our CSO, Jonas Korlach, will host the workshop, which includes:

* Increased Complexity of the Human Genome Revealed by Single-Molecule Sequencing
Evan Eichler, University of Washington 

* Defining a Personal, Allele-Specific, and Single-Molecule Long-Read Transcriptome
Hagen Tilgner, Stanford University

* Long-Read Multiplexed Amplicon Sequencing: Applications for Epigenetics and Pharmacogenetics
Stuart Scott, Icahn School of Medicine at Mount Sinai


Thursday, October 9, 2014

New Brain Study Reveals Higher Molecular Diversity from Alternative Splicing

A new paper from scientists in Switzerland and the US adds to recent findings about diversity of neuronal transcripts in the mammalian brain. The authors report that this study was only possible using long reads from Single Molecule, Real-Time (SMRT®) Sequencing.

Targeted Combinatorial Alternative Splicing Generates Brain Region-Specific Repertoires of Neurexins,” from lead author Dietmar Schreiner, senior author Peter Scheiffele, and collaborators, was published this month in the journal Neuron. The researchers are from the University of Basel, ETH Zurich, and North Carolina State University. This is the second study on neurexin mRNA diversity using PacBio® sequencing.


Monday, October 6, 2014

'The Quality of PacBio Data Is Beyond Compare': Eric Schadt on Applications of SMRT Sequencing to Human Genetics

As part of its continuing series on long-read sequencing, last week Mendelspod aired an engaging interview with Eric Schadt, Professor & Chair of Genetics and Genomic Sciences, and Director of the Icahn Institute for Genomics and Multiscale Biology at Mount Sinai.

Having now spent three years in his role at the groundbreaking institute, he reports that they are making great progress in the quest to build better data-driven health profiles around individuals that may better guide healthcare choices.

On short-read versus long-read sequencing
Short-read sequencing technologies still maintain the advantage in terms of throughput, says Schadt, but there are a variety of important genomic features that cannot be characterized without long-read sequencing, such as long tandem repeats, bigger structural variations, and focal variants important in cancer.


Thursday, October 2, 2014

‘We’re Going to Find the Keys’: Dan Geraghty Discusses an Approach to Understanding Causal Genetic Variation

Dan Geraghty, a researcher at Fred Hutchinson Cancer Research Center and CEO of Scisco Genetics, has spent much of his career focused on the genetics of immune response. Recently he talked to Mendelspod host Theral Timpson as part of a continuing series of podcasts on the rise of long-read sequencing.

Geraghty explained that while there have been decades’ worth of studies associating the genetics of the major histocompatibility complex (MHC), and the highly polymorphic HLA class 1 and 2 genes, we still haven’t found the key mutations for a variety of different autoimmune diseases such as type 1 diabetes, rheumatoid arthritis, multiple sclerosis, and others.

Enormous amounts of linkage disequilibrium in these regions are one factor, as is getting information in phase, so larger stretches of sequence are needed. Recently Geraghty has begun using Single Molecule, Real-Time (SMRT®) Technology with hopes of drilling down to the causal genetics.