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…
It was the coolest critter Erin Bernberg (@ErinBernberg) had ever worked with – quite literally. The senior scientist at the University of Delaware Sequencing and Genotyping Center, a PacBio certified service provider, received a shipment of tiny, live ice worms from Washington State University and immediately faced several challenges. How would she get them out of their ice cubes? How would she isolate DNA from the delicate, dark pigmented creatures? And would she be able to extract enough DNA to sequence? Thanks to the new PacBio low DNA input protocol, the answer to the last question was yes. In fact,…
Variety is the spice of life, and one of the drivers of genetic variation is gene splicing. After a gene is transcribed, there are alternatively spliced transcripts that add even more variety to that gene’s expression and its menu of phenotypes. It appears that there are types of disorders that take advantage of these varieties. Top amongst them are myeloid disorders, where somatic mutations in splicing factors lead to cell proliferation in myelodysplastic syndromes (MDS) and blood cancers. Christopher R. Cogle, a physician-scientist at the University of Florida, would like to understand why, in hopes that such knowledge could be…
Seeking to sequence and characterize entire transcriptomes in one go? Our new Iso-Seq protocol and reverse-transcriptase PCR kit makes it easier, speedier and cheaper. Run on the new Sequel II System, the completely revamped Iso-Seq Express workflow achieves whole transcriptome characterization from a single SMRT Cell 8M delivering up to 400 Gb, and at a third of the cost, or less. Yield has also increased on the Sequel System, with 3.0 sequencing chemistry typically delivering up to 30 Gb per SMRT Cell 1M for our RNA sequencing application. The new protocol requires three times less RNA input (300 ng) and…
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…
We’re thrilled to announce the launch of the Sequel II System, reducing project costs and timelines with approximately eight times the data output compared to the previous Sequel System. It enables customers to comprehensively detect human variants ranging in size from single nucleotide changes to large, complex structural variants. The system is also ideal for standard applications such as de novo assembly of large genomes and whole transcriptome analysis using the Iso-Seq method. The Sequel II System is based on the proven technology and workflow underlying the previous version of the system, but contains updated hardware to process the new…
For the thousands of scientists who attended The Plant and Animal Genome Conference in San Diego this January, the sentiment seemed to be “ask not if PacBio is for you, but how PacBio can work best for you.” The answer that emerged during PacBio’s PAG workshop and subsequent SMRT Informatics Developers Conference was a complex one. Recent developments, such as new chemistry, new SMRT Cells, the SMRTbell Express Template Prep Kit, and SMRT Link 6.0 software have already led to faster and easier library prep, longer reads with more data and reliability, better transcript characterization (Iso-Seq) and phasing (FALCON-Unzip) capabilities…
With their large brains, sophisticated sense organs and complex nervous systems, cephalopods could teach us a thing or two about learning, memory, and adaptability. But despite their evolutionary, biological, and economic significance, their genome information is still limited to a few species. To bridge this gap, a team of Korean scientists has assembled the genome of the common long-arm octopus (Octopus minor) using PacBio technology to sequence both the DNA and RNA of the emerging model species. Found in Northeast Asia, particularly in coastal mudflats of South Korea, China, and Japan, O. minor has become a major commercial fishery product…
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…
Today we’re pleased to announce the release of Sequel System 6.0, including new software, consumable reagents and a new SMRT Cell. Combined, the enhancements in the release improve the performance and affordability of Single Molecule, Real-Time (SMRT) Sequencing by providing individual long reads with greater than 99% accuracy, increasing the throughput up to 50 Gb per SMRT Cell, and delivering average read lengths up to 100,000 base pairs, depending on insert size. These improvements are expected to greatly enhance the accuracy and cost effectiveness of applications such as whole genome sequencing, human structural variant detection, targeted sequencing and RNA transcript isoform sequencing (Iso-Seq method). Estimated…