In this ASHG 2020 PacBio Workshop Emily Farrow of Children’s Mercy Kansas City shares how the incorporation of long-read sequencing into the Genomic Answers for Kids research study is increasing diagnostic yields through the identification of novel genetic variation. Emily highlights several cases in which PacBio HiFi sequencing was able to provide insights where short-read sequencing alone was inconclusive, due to limitations stemming from repetitive regions and large structural variants.
Most genes in eukaryotic organisms produce alternative isoforms, broadening the diversity of proteins and non-coding RNAs encoded by the genome. In contrast to other RNA sequencing platforms that rely on short-read sequencing, long accurate reads from PacBio Single Molecule, Real-Time (SMRT) Sequencing can characterize full-length transcripts without the need for assembly and inference. The PacBio isoform sequencing (Iso-Seq) method generates full-length sequences for transcripts up to 10 kb in length, with scalable throughput using barcoding approaches. The Iso-Seq application can be employed for a wide variety of studies, including improvement of gene annotation, identification of novel isoforms and fusion transcripts,…
Epigenetics expert Michael Jennings from Griffith University first posited the phasevarion, or the phase variable regulon mechanism in host-adapted pathogens. This mechanism switches expression of multiple genes in a coordinated fashion and has significant implications on pathogen virulence. In his talk, Jennings describes the phasevarion and his use of whole methylome data to rapidly identify methylation targets.
Shane Brubaker from renewable oil manufacturer Solazyme reports using the PacBio system to sequence the genome of a GC-rich strain of algae that couldn’t be fully assembled with short-read sequence data. He notes that CCS reads exceed Sanger quality at significantly lower cost.
In this BioConference Live webinar, PacBio CSO Jonas Korlach highlights how multi-kilobase reads from SMRT Sequencing can resolve many of the previously considered ‘difficult-to-sequence’ genomic regions. The long reads also allow phasing of the sequence information along the maternal and paternal alleles, demonstrated by full-length, fully phased HLA class I & II gene sequencing. In addition, characterizing the complex landscape of alternative gene products is currently very difficult with short-read sequencing technologies, and he describes how long-read, full-length mRNA sequencing can be used to describe the diversity of transcript isoforms, with no assembly required. Lastly, in the exciting area of…
At the PacBio ASHG workshop, Hagen Tilgner describes how he used long-read sequencing with Iso-Seq method to generate the first personal transcriptomes for three individuals. From these three family members, he and his collaborators were able to unambigously assign allele-specific RNA haplotypes, including HLA haplotypes, and demonstrated Mendelian inheritance of RNA molecules.
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.
Neema Mayor from Anthony Nolan Research Institute offers an introduction to the challenges of characterizing the HLA region, noting that improvements in resolution have allowed scientists to dramatically expand the number of classifications used to match donors to recipients. As sequencing resolution improves, Mayor says, scientists expect to find even more polymorphisms than what has been already catalogued.
In this webinar, the presenters describe a targeted sequencing workflow that combines Roche NimbleGen’s SeqCap EZ enrichment technology with PacBio’ SMRT Sequencing to provide a more comprehensive view of variants and haplotype information over multi-kilobase, contiguous regions. They demonstrate that 6 kb fragments can also be utilized to enrich for long fragments that extend beyond the targeted capture site and well into (and often across) the adjacent intronic regions. When combined with SMRT Sequencing, multi-kilobase genomic regions can be phased and variants, including complex structural variants, can be detected in exons, introns and intergenic regions.