Yunfei Guo, from the University of Southern California, presents his ASHG 2015 poster on a de novo assembly of a diploid Asian genome. The uniform coverage of long-read sequencing helped access regions previously unresolvable due to high GC bias or long repeats. The assembly allowed scientists to fill some 400 gaps in the latest human reference genome, including some as long as 50 kb.
Alex Dainis, a graduate student in Euan Ashley’s lab at Stanford University, presents her ASHG 2015 poster on haplotyping for genes linked to hypertrophic cardiomyopathy. Using the Iso-Seq method with SMRT Sequencing, she sequenced full transcripts of two genes of interest, generating data on 150 different isoforms. Rare variants, which could not be found with other technologies, were associated with haplotypes.
Masao Nagasaki from Tohoku University presents in his ASHG 2015 poster on typing of HLA class I genes using SMRT Sequencing. By using long-read sequencing he was able to successfully type these genes for 220 individuals. This included samples that he had previously been unsuccessful typing using short-read sequencing.
Computational biologist Mauricio Carneiro, PhD, describes a Broad Institute technology comparison to determine how PacBio, Ion Torrent, and Illumina MiSeq perform in discovering and validating human SNPs. Noted PacBio advantages: no bias in GC regions, no systematic errors, and no sequence degradation over increased read length. In a study using samples from the 1,000 Genomes project, PacBio outperformed MiSeq and Ion Torrent in sensitivity and specificity.
Tyson Clark, a scientist at PacBio, demonstrates the detection and identification of damaged DNA using SMRT Sequencing. With the platform's ability to see base modifications, Clark notes that the polymerase kinetics can distinguish between different types of DNA damage as well — such as oxidative, radiation, and alkylation. This could help in studies of cancer and aging, where DNA damage is an important factor.
Anne Deslattes Mays from Georgetown University presents her AGBT poster on the balancing act of discovering transcriptome isoforms. Using SMRT Sequencing to study differentiated and undifferentiated cells from human bone marrow, she analyzed full-length isoforms and confirmed unexpected findings with mass spec. She says access to unfragmented long reads allows scientists to move from transcripts to proteins.
Ulf Gyllensten from Uppsala University describes his AGBT poster showing the use of SMRT Sequencing for HLA allele typing. He says long reads are essential for sequencing the HLA genes because they link exons in a single read and do not introduce bias, as short-read sequencers can. Looking at fusion transcripts from CML patients generated information that couldn't be achieved with any other technology, he adds.
Jonas Korlach, of PacBio, discusses the use of SMRT sequencing to detect DNA modifications.
Ellen Paxinos, a scientist at PacBio, shares her AGBT poster on work done in collaboration with reference lab Monogram Biosciences using Single Molecule, Real-Time (SMRT) sequencing to detect minor species and variants in HCV. Using two genotypes mixed together, the team was able to detect variants down to 1% and to identify both viral haplotypes from the data. Paxinos says the study is a model for looking at genomic variation in chronic viral infection.
Dan Geraghty from the Fred Hutchinson Cancer Research Center presents his AGBT poster on a new PacBio-based solution to sequence extended genomic regions — in this case, KIR and MHC, two of the most variable regions of the human genome. He reports data revealing for the first time regions that may be associated with autoimmune diseases such as diabetes, rheumatoid arthritis, and multiple sclerosis, and also shows that sequences were phased, complete, and highly accurate.
Yuta Suzuki from the University of Tokyo presents his AGBT poster on heterozygotic DNA methylation patterns. He used kinetic data from SMRT Sequencing to generate epigenetic information on samples ranging from human to medaka fish and was able to analyze haplotype-specific methylation data. He also shows that long reads are better able to capture data about CpG islands than short-read sequences.
PacBio scientist Khai Luong reviews her AGBT poster which was the first example showing SMRT Sequencing could be used to directly detect epigenetic modification in a eukaryotic organism.
PacBio scientist Ellen Paxinos discusses a study presented at AGBT that gnerated single-molecule full genome sequencing of HIV 1 from two pairs of linked transmission from a Zambian cohort. Sequencing was done on full-length amplicons from the virus, and clustering accurately placed the virus from each pair together, distinguishing between the two pairs. Paxinos notes that 50 MB of sequence data was generated in less than four hours.
Penelope Bonnen, an assistant professor at Baylor College of Medicine, discusses her use of PacBio SMRT sequencing to look at whole mitochondrial genomes as she reviews her AGBT 2012 poster. Dr. Bonnen is studying a Micronesian population with unusually high rates of obesity, diabetes, and cardiovascular disease to figure out how mitochondrial genetics contributes to adult-onset metabolic syndrome. She describes two approaches in a pilot project for full-length mitochondrial sequencing: one using a 500-base pair insert library and another directly sequencing the single 17 kb amplicon.
PacBio scientist Cheryl Heiner describes new low-input protocols for SMRT Sequencing library construction. With these revised methods, 2 kb libraries can be generated from as little as 10 ng of DNA, while 10 kb libraries require only 100 ng of sample.