PacBio Sequencing is characterized by very long sequence reads (averaging > 10,000 bases), lack of GC-bias, and high consensus accuracy. These features have allowed the method to provide a new gold standard in de novo genome assemblies, producing highly contiguous (contig N50 > 1 Mb) and accurate (> QV 50) genome assemblies. We will briefly describe the technology and then highlight the full workflow, from sample preparation through sequencing to data analysis, on examples of insect genome assemblies, and illustrate the difference these high-quality genomes represent with regard to biological insights, compared to fragmented draft assemblies generated by short-read sequencing.
Rebecca Johnson, director of the Australian Museum Research Institute presents finding from de novo sequencing of the koala genome. Using PacBio sequencing the Koala Genome Consortium obtained an assembly with an N50 of 11.5 Mbp and have undertaken functional genomic analysis highlighting the unique genes associated with lactation and immune function of koalas. Johnson goes on to describe efforts to obtain a chromosome level assembly and current work using ‘super scaffolding’ to compare shared synteny across diverse lineages to generate chromosome scaffold maps.
In this AGBT 2017 poster, Ulf Gyllensten from Uppsala University presents two local reference genomes generated with PacBio and Bionano Genomics data. These assemblies include structural variation and repetitive regions that have been missed with previous short-read efforts, including some new genes not annotated in the human reference genome.
In this Webinar, we will give an introduction to Pacific Biosciences’ single molecule, real-time (SMRT) sequencing. After showing how the system works, we will discuss the main features of the technology with an emphasis on the difference between systematic error and random error and how SMRT sequencing produces better consensus accuracy than other systems. Following this, we will discuss several ground-breaking discoveries in medical science that were made possible by the longs reads and high accuracy of SMRT Sequencing.
At AGBT 2017, the Broad Institute’s Daniel Neafsey reported a large collaborative effort to sequence the mosquito that carries Zika virus. The team is using long-read PacBio sequencing to produce a high-quality genome assembly, which Neafsey expects will replace the 10-year-old Sanger assembly for Aedes aegypti. The new assembly reduces the number of contigs by at least 10-fold, boosts the contig N50 to nearly 2 Mb, and features more complete gene content.
At AGBT 2017, Margaret Roy from Calico Life Sciences discussed a de novo genome sequencing effort for the naked mole rat. This animal has a remarkably long life span and resistance to cancer, both of which make it interesting for studies of life extension. The team is using SMRT Sequencing for a more complete, contiguous assembly than the two existing short-read-based assemblies. Included: data from the Sequel System.
At AGBT 2017, Mike Schatz from Johns Hopkins University and Cold Spring Harbor Laboratory presented data from sequencing, assembling, and analyzing personalized, phased diploid genomes with either Illumina, 10x Genomics, and PacBio SMRT Sequencing. Compared to the short-read-based methods, PacBio data assembled in large, complete contigs and contained the broadest range of structural variants with the best resolution. Plus: unexpected translocation findings with SMRT Sequencing, validated in follow-up studies.
In this webinar, Emily Hatas of PacBio shares information about the applications and benefits of SMRT Sequencing in plant and animal biology, agriculture, and industrial research fields. This session contains an overview of several applications: whole-genome sequencing for de novo assembly; transcript isoform sequencing (Iso-Seq) method for genome annotation; targeted sequencing solutions; and metagenomics and microbial interactions. High-level workflows and best practices are discussed for key applications.
To make improvements to crops like corn, soybeans, and canola, scientists at Corteva are building a compendium of crop genomics resources to provide actionable sequence info for genetic discovery, gene-editing, and seed product development. Hear how Kevin Fengler, Comparative Genomics Lead of Data Science and Bioinformatics at Corteva, is using PacBio sequences to build visualization tools and genome assembly pipelines as a contribution to this effort.
In this presentation, Sonja Vernes of the Max Plank Institute shares her work with the Bat1K project which aims to catalog the genetic diversity of all living bat species. She highlights the unique biology of bats, from their widely varying sizes to their capacity for healthy aging and disease resistance and provides recent findings from ongoing efforts to sequence and annotate the genomes of 21 phylogenetic families of bats.
To start Day 1 of the PacBio User Group Meeting, Jonas Korlach, PacBio CSO, provides an update on the latest releases and performance metrics for the Sequel II System. The longest reads generated on this system with the SMRT Cell 8M now go beyond 175,000 bases, while maintaining extremely high accuracy. HiFi mode, for example, uses circular consensus sequencing to achieve accuracy of Q40 or even Q50.
In this PacBio User Group Meeting presentation, Nic Wheeler of University of Wisconsin-Madison, speaks about RNA sequencing for filarial nematodes associated with understudied tropical diseases. His team used Iso-Seq analysis to improve gene models and achieve better transcriptome coverage for these worms, which typically have poorly annotated and fragmented genome assemblies. While getting enough RNA to study is a technical challenge, the group still managed to generate full-length isoforms, many of which were novel or contained novel junctions.
In a push to develop insect-based food sources for people, Brenda Oppert from the USDA has been sequencing bug genomes with PacBio technology. Long reads are essential because of the highly repetitive sequences and large genomes. On the Sequel II System, a single SMRT Cell is sufficient to generate 350-fold coverage and produce a high-quality assembly for some of the insects she’s studying.
In this PacBio User Group Meeting presentation, Erin Bernberg from the University of Delaware reports on using the Agilent Femto Pulse System for high-resolution, highly sensitive fragment analysis and on the low DNA input protocol, which her team used for a recent study of ice worms.
Tina Graves-Lindsay from the McDonnell Genome Institute reports at AGBT 2020 on how her team is using PacBio sequencing to produce reference-grade human genome assemblies. With highly accurate HiFi reads, no error correction step is needed during the sequencing and analysis process, and they can produce reference-grade assemblies with half the sequence coverage needed before. They are now generating diploid assemblies and will be contributing to the human pangenome reference project.