About two months back, we reported on an user group meeting arranged by Pacific Biosciences.
Those looking for the actual talk abstracts and slides will find the following links helpful. We thank PacBio for providing them.
Alex Copeland, DOE - Joint Genome Institute
Copeland offered an overview of the institutes microbial and fungal reference assembly pipeline, describing their experience with a 10x increase in read length and total throughput in three years on the PacBio platform. He shared that the team has shifted to a PacBio-only pipeline, and that they are finishing genomes on the platform for less than $2,000.
Matthew Blow, DOE - Joint Genome Institute
Blow spoke about bacterial epigenomics, an important genome component that his team looks at with every microbe sequenced. The team is studying methyltransferases, their link to restriction enzymes, related sequence motifs, and sites that are unmodified. A recent analysis of global patterns in DNA modifications in bacteria revealed that of 198 analyzed genomes, 169 (>90%) had modified DNA bases.
Bart Weimer, UC Davis - School of Veterinary Medicine
Weimer spoke about the 100K Foodborne Pathogen Genomes project and its goal to provide a comprehensive database that will allow users to find clinically relevant information about new strains in outbreak situations. He was enthusiastic about the additional information provided by PacBio sequence data, such as methylation and phage elements both useful in tracking and identifying pathogens.
Lance Hepler, Center for AIDS Research, UC San Diego
Hepler used the PacBio RS to study intra-host diversity in HIV-1. He compared PacBios performance to that of 454, the platform he and his team previously used. Hepler noted that in general, there was strong agreement between the platforms; where results differed, he said that PacBio data had significantly better reproducibility and accuracy.
George Weinstock, Washington University St. Louis
Weinstock discussed his overall approach to human microbiome projects, including both targeted 16S sequencing with PacBio, as well as shotgun sequencing of the whole sample. In a pilot project, Weinstocks team created a mock microbiome of 24 samples with a 300-fold range of concentration; PacBio sequencing was able to accurately identify the taxa for all 22 species where 16S amplification succeeded, yielding highly accurate full-length 16S consensus sequences.
John Huddleston, University of Washington
Huddleston is looking at challenging regions in the human genome, noting
that assembly accuracy needs to be quite high to resolve breakpoints and
reconstruct duplication architectures. His team is working with BACs to
validate the use of the PacBio platform as a faster, more cost-effective
alternative to Sanger. In one study, his team found that PacBio results
had 99.994% identity with Sanger results and showed uniform coverage
across the clone.
Lisbeth Guethlein, Stanford University School of Medicine
Guethlein looked at highly repetitive and variable regions of the
orangutan genome. Guethlein reported that PacBio managed to accomplish
in a week what I have been working on for a couple years, (with
Sanger) and the results were concordant.
Vince Magrini, Washington University St. Louis
Magrini described a proof-of-principle RNA-seq study using SMRT
Sequencing in a nematode to help elucidate transcriptional regulation and
its effect on life cycle. Using PacBio data added more than 1,500 genes
to what had been found in the reference sequence.
Alisha K. Holloway, Gladstone Institute
Holloway presented data from transcript identification work in chicken.
Because she uses chicken to model human heart development, she needs good
annotations of RNA produced at various developmental stages to figure out
where problems arise. Unlike short-read technologies, PacBio provided
reads long enough to span entire transcripts and dramatically improved
Bobby Sebra, Mt. Sinai School of Medicine
Sebra showed how to use BluePippin size selection from Sage Science to increase subread lengths of PacBio data. He noted that the BluePippin sizing step also cleans up DNA quality, compensating for any drop in yield. With size selection, Sebra said that his team could generate microbial assemblies from data produced by a single SMRT Cell.
Kin Fai Au, Stanford University
(slides not currently available)
Au spoke about gene isoform identification and prediction in embryonic stem cells, commenting that long reads are essential to examining these long regions and resolving alternative splice isoforms.
Chongyuan Luo, Salk Institute for Biological Studies
(slides not currently available)
Luo from the Ecker lab spoke about studying the genome and epigenome of several Arabidopsis thaliana strains using SMRT Sequencing. PacBio sequence data detected 40 percent more SNPs than short-read technology, indicating that some regions may not have been covered well enough with short reads to find all SNPs.
Kevin Corcoran, Pacific Biosciences
Corcoran updated attendees about the latest on our sequencing platform, including coming advances such as polymerase photodamage protection, the new P5-C3 chemistry offering 8,500 base average reads, three-hour movies, Quiver for diploid sequencing, and more.
Jonas Korlach, Pacific Biosciences
Korlach spoke about where the PacBio platform is heading, including use for customer projects that include large numbers of samples, higher complexity metagenomic studies, and assemblies of larger genomes. He also mentioned upcoming technology improvements, such as library prep automation and new data analysis algorithms.