Traditional RNA-Seq is done by fragmenting cDNA, and then sequencing the fragmented reads with paired-end sequencing. The problem comes when trying to identify the full-length isoform during assembly. This is computationally challenging, and sometimes intractable. Iso-Seq is a method of full-length transcript sequencing that eliminates the need for assembly The solution? Long-read isoform sequencing, according to PacBio Principal Scientist Elizabeth Tseng and PacBio user Gloria Sheynkman, a research fellow at Dana-Farber Cancer Institute. The two recently participated in a webinar, sharing their experiences using PacBio’s Iso-Seq method. Tseng started by explaining the method and some of its applications. “In contrast…
At ASHG 2019, PacBio scientists Aaron Wenger and Liz Tseng offered a CoLab presentation. At the annual meeting of the American Society of Human Genetics in Houston, PacBio scientists presented how our Sequel II System performs for structural variant (SV) detection and for whole transcriptome sequencing. The educational workshop focused on experiments that can be done using a single SMRT Cell 8M on the Sequel II System. The event kicked off with Aaron Wenger walking through SV analysis, which he said has mirrored the development path of single nucleotide variants, from proof-of-concept to individual rare disease studies and now to…
When looking to understand the functional implications of genetic variability, scientists should seek out the Iso-Seq method, according to Cold Spring Harbor researchers. In a recent paper published in Frontiers in Genetics, Doreen Ware, Bo Wang, and colleagues reviewed the state of transcript sequencing and analysis technologies, and concluded that single-molecule sequencing from PacBio provided several advantages over other methods. A major challenge in molecular biology continues to be the complex mapping of the same genome to diverse phenotypes in different tissue types, development stages and environmental conditions, the paper states. “A better understanding of the transcripts and expression of…
One of the fastest growing global foods is also one of its most vulnerable. Without an adaptive immune system, the Pacific white shrimp, Litopenaeus vannamei, rely on cellular and humoral defenses, such as the release of antimicrobial peptides, in their battle against invading microbes and pathogen infections. A battle they’re losing, leading to massive mortality and devastating economic losses. A full-length transcriptome analysis using the PacBio Iso-Seq method has resulted in an isoform-level reference transcriptome that is shedding new light into the shrimp’s innate immune system, providing hope for the shrimp aquaculture industry. One of the most economically important shrimp…
Genome-wide association studies (GWAS) may be powerful tools for the identification of genes underlying complex traits, but what if you have an incredibly complex, uncharacterized genome, with no sequenced progenitor or related species? A team of scientists from the Chinese Academy of Agricultural Sciences in Changsha, China came up with a solution: a transcriptome-referenced association study (TRAS), powered by our Iso-Seq method. The approach, outlined in this DNA Research paper, utilized a transcriptome generated by SMRT Sequencing as a reference to score population variation at both transcript sequence and expression levels. The team, led by Touming Liu and first author Xiaojun…
A paper from scientists at Colorado State University and the National Center for Genome Resources provides an in-depth view of the transcriptome of sorghum, a crop that’s important for human and animal food and also shows potential as a biofuel. Through this project, the team produced a new isoform analysis pipeline for community use and identified novel genes, as well as far more alternative splicing than had been expected for this plant. The publication, “A survey of the sorghum transcriptome using single-molecule long reads,” comes from lead author Salah Abdel-Ghany, senior author Anireddy Reddy, and collaborators. The researchers were particularly…
In a new publication from Cold Spring Harbor Laboratory, scientists produced a dataset for what authors call “the single largest collection of [full-length] cDNAs available in maize” and significantly improved genome annotation. The effort relied on the Iso-Seq method with SMRT Sequencing, which allows scientists to generate ultra-long reads covering full transcripts. The paper, “Unveiling the complexity of the maize transcriptome by single-molecule long-read sequencing,” comes from lead author Bo Wang and senior author Doreen Ware, who is also affiliated with the USDA Agricultural Research Service. It offers the first published results from using the Iso-Seq method on a maize…