Photo by John Cobb on Unsplash In eukaryotic organisms, the majority of genes are alternatively spliced to produce multiple transcript isoforms. Gene regulation through alternative splicing can dramatically increase the protein-coding potential of a genome. Therefore, understanding the functional biology of a genome requires knowing the full complement of isoforms. Microarrays and high-throughput cDNA sequencing are useful tools for studying transcriptomes, yet these technologies provide only small snippets of transcripts. Accurately reconstructing complete transcripts to study gene isoforms has been challenging [1, 2]. The Iso-Seq method produces full-length transcripts using Single Molecule, Real-Time (SMRT) Sequencing [3]. Long read lengths enable…
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…
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…
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…
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…
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…