A series of recent peer-reviewed scientific publications highlights the growing use of Single Molecule, Real-Time (SMRT®) Sequencing to annotate plant and animal genomes. These reports from the genomics community show that PacBio sequencing consistently detects more genes, alternative splicing events, and other important elements compared with competing sequencing platforms. Characterizing these elements is essential for a deeper understanding of these organisms as well as for breeding efforts to improve crops and livestock.
Genome annotation using short-read technology (RNA-seq) is challenging because individual reads are too short to span entire gene transcripts, which can be multiple kilobases long. As a result, scientists must stitch together many short reads using error-prone assembly processes. Alternative splicing, in which the same gene expresses functionally different transcripts, is particularly difficult to detect with short reads because segments of those transcripts are often conflated during assembly. SMRT Sequencing produces significantly longer reads, achieving mean lengths of 10-18 kb, allowing scientists to generate full-length transcripts or isoforms without introducing errors from assembly algorithms. For transcriptome analysis, this is known as the Iso-Seq™ method because it produces full-length isoform sequences.
“Scientists are embracing the Iso-Seq method to produce a more complete genetic view of everything from important crops, such as wheat and barley, to livestock, including chickens,” said Kevin Corcoran, Senior Vice President of Market Development for Pacific Biosciences. “The beauty of this approach is that SMRT Sequencing provides a lot of novel information about gene content even for organisms that lack a reference assembly or have incredibly complex genomes. With our latest instrument, the Sequel System, we anticipate that more groups will adopt SMRT Sequencing as a fast and affordable means of generating the most comprehensive transcriptomes possible.”
“In our analysis of gene activity in chickens, SMRT Sequencing allowed us to overcome challenges that had limited our view of genes in this bird for years,” said David Burt, Professor at the University of Queensland, Genomics Initiative, who serves on the steering committee for the Functional Annotation of Animal Genomes (FAANG) Project. “A major advance over previous methods was the accurate characterization of all alternative splice transcripts – from start to end – allowing us to define all transcription start sites. More importantly, for the first time we were able to define accurate long non-coding RNAs. Based on this success, we anticipate implementing the Iso-Seq method for transcriptomic studies of many livestock species across the FAANG consortium as the surest path to fully characterizing the gene content in these animals.”
Recent Iso-Seq publications include:
Normalized long read RNA sequencing in chicken reveals transcriptome complexity similar to human, BMC Genomics
Principal investigator David Burt and his team studied embryonic and brain tissue from chickens, finding 70% more genes than were previously known to exist in this animal.
Led by Matthew Clark, this project analyzed gene content in Chinese Spring bread wheat. PacBio data revealed more than 6,600 genes missed by other technologies, and corrected more than 8,100 gene models that were inaccurate in previous annotations.
Principal investigator Nils Stein and collaborators used SMRT Sequencing and other tools to characterize the transcriptome of barley. Their analysis revealed about 40,000 high-confidence genes.
Senior author Young Chang Sohn and team reported a detailed analysis of gene content in abalone, finding that transcriptome patterns differed substantially between males and females. More than 500 genes in females and nearly 400 genes in males had multiple isoforms, i.e., evidence of alternative splicing.
Single-cell mRNA isoform diversity in the mouse brain, BMC Genomics
Scientists Kasper Karlsson and Sten Linnarsson report unexpected alternative splicing diversity in mouse brain tissue, with many cells expressing unique isoforms.
For more information, please visit http://www.pacb.com/research-focus/plant-animal-sciences/.
Source – Globe Newswire