PacBio Blog

Tuesday, December 16, 2014

At ASHI 2014, SMRT Sequencing Meets HLA Typing with Great Results

Earlier this fall, we headed to Denver for ASHI, or the annual meeting of the American Society for Histocompatibility and Immunogenetics. Though we’d attended this conference in the past, this was our first year having an exhibit hall booth and workshop, both of which were enthusiastically received by the conference attendees. Even though it’s a fairly recent development for scientists to apply the PacBio® sequencing platform to analyze the HLA genes, which are often used in histocompatibility research studies, there were already many great examples and exciting data generated by users on the PacBio platform.

Our luncheon workshop on fully phased HLA and KIR typing was packed, and that was no doubt due to our top-tier speakers: Prof Steve Marsh, from the Anthony Nolan Research Institute and University College London; Nezih Cereb, CEO & Co-Founder of Histogenetics; and Martin Maiers, Director of Bioinformatics Research at the National Bone Marrow Donor Program. We were able to record video of their presentations, which you can check out below. You can also peruse some posters showcasing Single Molecule, Real-Time (SMRT®) Sequencing for HLA and KIR analysis, as well as a webinar recorded at the meeting.

Workshop presentations

Steve Marsh spoke about the challenges of HLA typing in a clinical laboratory and his vision for a more holistic approach to HLA gene sequencing. He also called for submissions of only full-length, phased HLA sequences to the IMGT/HLA reference database.

Nezih Cereb’s talk focused on the addition of SMRT Sequencing to the research arsenal at Histogenetics; they were the first HLA typing lab in the world to acquire a PacBio instrument. In the presentation, he discussed their efforts to enhance the resolution and accuracy of HLA typing. Recording will be available soon.

Martin Maiers spoke about the use of KIR genomic content for clinical transplantation, as well as outcomes of HLA sequencing from a variety of methods. He also shared recent data from experiments with PacBio sequencing of fosmid libraries.

In an introduction to the workshop, PacBio’s Swati Ranade offered a primer on the use of SMRT Sequencing for full-length, phased, imputation-free HLA gene analysis with allele-level typing. She also spoke about the new PacBio chemistry, which provides longer reads to enhance HLA and KIR analysis.

In addition, we recorded the workshop Q&A.

Customer testimonial

Dr. Nezih Cereb, CEO & co-founder of HistoGenetics shares his reasons for adopting the PacBio DNA Sequencing platform into their operations, and elaborates on the PacBio RS II System’s unique ability to sequence full-length HLA genes and to provide fully phased HLA alleles.


Application of Single Molecule Real-Time (SMRT) Sequencing Technology for the Field 4 Level Genotyping of Classical HLA Loci

Evaluation of Multiplexing Strategies for HLA Genotyping Using PacBio® Sequencing Technologies

View additional HLA resources at

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 tasks like de novo discovery of genes, gene isoforms assembly or transcript and isoform abundance determination are still challenging and far from being achieved.”

They report on a previous paper from Au in which Single Molecule, Real-Time (SMRT®) Sequencing was combined with short-read sequencing to detect isoforms in a well-characterized human embryonic stem cell line. Long reads led to the detection of hundreds of novel isoforms and long noncoding RNAs. Long intergenic noncoding RNAs (lincRNAs) are a topic of interest in the review article, where Au and Sebastiano note that they “have a very high degree of repetitive elements and it is therefore extremely challenging to determine the correct gene annotation and the abundance due to the difficulties in aligning short read data to the genome.” With long-read sequencing, they add, sequence data spans unique sections of the lincRNAs and makes it possible to accurately map reads to the correct region.

The authors cite recent studies demonstrating more transcriptional activity in the human genome than has been expected. “Transcription occurs across 80–90% of the human genome, in contrast with the assumption that only 3% (or less) of the genome is actually coding for proteins,” they write. LincRNAs and other noncoding RNAs may explain the difference between those numbers.

Au and Sebastiano call for more studies of stem cells using long-read sequencing technology to establish a better view of the transcriptional activity in these important cells with accurate detection of noncoding RNAs characterized by highly repetitive sequence. “Given such complexity of the epigenetic status for most of the genes, it is essential to identify the transcripts and the isoforms that are indeed functionally relevant (even if expressed at low levels) in [pluripotent stem cells],” they write.

Thursday, December 4, 2014

A New Reference Genome for Shigella: SMRT Sequencing of a Historic Sample

In a special issue of The Lancet dedicated to World War I, an article by scientists from the Wellcome Trust Sanger Institute used Single Molecule, Real-Time (SMRT®) Sequencing to decode the genome of the first isolate ever collected of Shigella flexneri.

The bacterium, a descendant of E. coli and first identified as a separate strain in 1902, was responsible for severe dysentery among World War I troops due to poor hygienic conditions in the trenches. Today, S. flexneri is one of the leading causes of diarrheal death among children in developing countries and other areas of poor sanitation.

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.

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