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Thursday, December 11, 2014

Review Article: Long-Read Sequencing Offers Better Understanding of Pluripotency

A new review article offers a nice overview of attempts to characterize the transcriptome of human stem cells using RNA-seq, the Iso-Seq™ method, and more. Kin Fai Au and Vittorio Sebastiano, scientists at the University of Iowa and Stanford University, respectively, contributed the review to Current Opinion in Genetics & Development. “The introduction of the RNA-Seq technology based on [second-generation sequencing technology] has provided a remarkable step forward providing a fast and inexpensive way to determine the transcriptome of a given cell type and several remarkable works have been done using this type of approach,” Au and Sebastiano write. “Nonetheless…

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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…

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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…

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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…

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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. The team tackled…

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Thursday, September 11, 2014

The Rise of Long Reads: Mendelspod Podcast Series

Mendelspod host Theral Timpson kicked off a new podcast series this week on long-read sequencing that will include interviews with luminaries in the genomics field. Check out this introductory article from Timpson for an explanation of why scientists are demanding longer reads to meet their research goals. The first interview is with Mike Snyder at Stanford, who has published recent papers in Nature Biotechnology and PNAS using Single Molecule, Real-Time (SMRT®) Sequencing for transcriptome analysis and demonstrated that long reads enable full coverage of RNA molecules. He discusses that work and his views on long-read sequencing and transcriptomics on the…

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Tuesday, July 1, 2014

Scientists Generate the First Personal Transcriptome Using SMRT Sequencing

A new paper from scientists at Stanford University and Yale University describes the use of Single Molecule, Real-Time (SMRT®) Sequencing to generate transcriptomes for three individuals. The work is believed to be the first personal transcriptome analysis using long-read sequencing. The paper, entitled “Defining a personal, allele-specific, and single-molecule long-read transcriptome,” was published in PNAS by Hagen Tilgner, Fabian Grubert, Donald Sharon, and Michael Snyder. Last year, the same authors published a study using SMRT Sequencing to analyze transcriptomes across tissue samples from human organs. In the PNAS publication, they compare metrics from the new data set to those from…

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Monday, June 2, 2014

ARCHIVED: Intro to the Iso-Seq Method: Full-length transcript sequencing

Since the launch of the Iso-Seq protocol in SMRT Analysis in 2014, the analysis pipeline has seen several improvements, as well a host of new tools. Please see our updated post to learn more about current applications of this increasingly popular method. With the recent launch of SMRT Analysis v2.2, we’re excited to introduce analysis software support for the new Iso-Seq™ method for sequencing full-length transcripts and gene isoforms, with no assembly required! Today we’ll take a deeper look at the Iso-Seq method to explain its unique scientific value and review publications from those already applying Single Molecule, Real-Time (SMRT®)…

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Wednesday, May 28, 2014

The Sequence Analysis Meeting: SFAF 2014

The Sequencing, Finishing, and Analysis in the Future (SFAF) meeting kicks off today in Santa Fe, New Mexico. The conference is hosted by Los Alamos National Laboratory and focuses on the analytical details that are so important as the community assesses how to get the most out of all this sequence data. This year, we will have two PacBio speakers, and there will be a number of other talks from users of our long-read sequence data. Steve Turner, our CTO, will speak on Wednesday morning about the use of Single Molecule, Real-Time (SMRT®) Sequencing for generating highly contiguous genome assemblies…

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Monday, May 5, 2014

Webinar Recap: New Insights in Genome and Transcriptome Research

This week, our CSO Jonas Korlach hosted a webinar entitled “Gain New Insights in Genome and Transcriptome Research with Greater than 10,000 bp Reads.” He spoke to attendees about the PacBio® technology, elements of sequencing, and applications of the ultra-long reads generated by Single Molecule, Real-Time (SMRT®) Sequencing. Here’s a quick recap. Jonas offered a look at how PacBio’s technology performs in the four key sequencing characteristics that one should consider for any sequencing work: contiguity, accuracy, uniformity, and originality. For contiguity, or how much of a DNA fragment can be sequenced in a single pass, the PacBio platform outperforms…

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Thursday, April 24, 2014

SMRT Sequencing of Chicken Heart Transcripts Yields New Genes and Isoforms

The Gallus gallus (common chicken) genome was initially published in 2004, but the latest RefSeq and Ensembl annotations remain incomplete. The chicken is an important model organism, especially for research on embryogenesis and heart development. In a new paper published in PLOS One, researchers representing the Cardiovascular Development Consortium of the Bench to Bassinet Program and Pacific Biosciences describe work to improve the chicken genome annotation using SMRT® DNA Sequencing. In “Long-Read Sequencing of Chicken Transcripts and Identification of New Transcript Isoforms,” the consortium describes how they used SMRT sequencing to generate full-length cDNA reads from embryonic chicken hearts, combined…

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Monday, March 31, 2014

New Software Release: SMRT Analysis Upgrade for Iso-Seq Analysis and HLA Allele Phasing

Today we announced the release of a software upgrade for our Single Molecule, Real-Time (SMRT®) DNA Sequencing platform. SMRT Analysis 2.2 provides enhanced functionality to support two additional applications that uniquely benefit from our long-read sequencing technology: Iso-Seq™ full-length transcript /isoform sequencing, and human leukocyte antigen (HLA) allele phasing. The study of mRNA transcript isoforms has been challenging due to the short read lengths of other sequencing technologies. Since Iso-Seq analysis allows for the capture of full-length transcripts, scientists can use this approach to identify alternatively spliced forms of a gene, detect novel genes and isoforms, or perform transcriptome-wide analysis…

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Friday, March 21, 2014

ABRF Preview: Off to the Land of Core Labs and Hot Air Balloons

Members of the PacBio team will be heading down to Albuquerque this weekend for the annual conference of the Association of Biomolecular Resource Facilities (ABRF). It’s a big year for the association, which is celebrating its 25th anniversary, and we’re looking forward to participating. ABRF is a meeting where we connect with the most innovative leaders of the core facility community. As PacBio blog readers know, core labs are very important in the SMRT® Sequencing landscape. Last year we launched our directory of service providers that offer sequencing with the PacBio® platform, and many scientists have told us how useful…

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Wednesday, March 19, 2014

Assessment of Highly Complex Alternative Splicing of Neurexins Performed with SMRT Sequencing

A new paper in the Proceedings of the National Academy of Sciences from the laboratories of Stephen R. Quake and Thomas C. Südhof (both at Stanford University) describes the direct, full-length transcript sequencing of RNA molecules that are essential to synapse formation in the mammalian brain. The team used Single Molecule, Real-Time (SMRT®) Sequencing to analyze full-length mRNAs from different members of the neurexin gene family and used that information to examine alternative splicing events. In the publication entitled “Cartography of neurexin alternative splicing mapped by single-molecule long-read mRNA sequencing,” the scientists highlight the importance of understanding alternative splicing in…

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Friday, March 14, 2014

AGBT 2014 Presentation Videos: SMRT Sequencing at CSHL, Uppsala U., and Baylor College of Medicine

There were several excellent talks showcasing SMRT® Sequencing data at the annual Advances in Genome Biology and Technology conference. If you didn’t have the opportunity to see them in person, you can watch the recordings: From Cold Spring Harbor Laboratory, Dick McCombie described the need for de novo sequencing, which preserves structural information that can be missed with resequencing. Organisms presented include yeast, Arabidopsis, and rice. McCombie notes that in many cases, full chromosomes are assembled into single contigs with long-read sequencing. He also presented the longest read seen at AGBT: more than 54 Kb. Watch video: A near perfect…

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