As the foundation for scientific discoveries in genetic diversity, sequencing data must be accurate and complete. With highly accurate long-read sequencing, or HiFi sequencing, there is no longer a compromise between read length and accuracy. HiFi sequencing enables some of the highest quality de novo genome assemblies available today as well as comprehensive variant detection in human samples. PacBio HiFi libraries constructed using our standard library workflows require at least 3 µg of DNA input per 1 Gb of genome length, or ~10 µg for a human sample. For some samples it is not possible to extract this amount of…
From crop improvement to breeding healthier livestock to modeling human disease, scientists are using PacBio Sequencing to advance understanding of plant and animal genomes. In this article, we look at four examples of plant and animal genome references improved or made possible with SMRT Sequencing, including an early example of transcriptome sequencing of a chicken for improved annotation. These examples highlight insights gained with SMRT Sequencing that are missed with short-read data, such as complex regions or novel genes.
At the National Center for Genome Resources in Santa Fe, New Mexico, scientists run a world- renowned sequencing service facility that’s heavy on long reads and bioinformatics expertise. It also supports a wide range of microbial, plant, and animal projects.
The SMRTbell Express Template Prep Kit 2.0 provides a streamlined, single-tube reaction strategy to generate SMRTbell libraries from 500 bp to >50 kb insert size targets to support large-insert genomic libraries, multiplexed microbial genomes and amplicon sequencing. With this new formulation, we have increased both the yield and efficiency of SMRTbell library preparation for SMRT Sequencing while further minimizing handling-induced DNA damage to retain the integrity of genomic DNA (gDNA). This product note highlights the key benefits, performance, and resources available for supporting de novo genome sequencing and structural variant detection projects. Our large-insert gDNA protocol has been streamlined to…
In this LabRoots webinar, Jonas Korlach the CSO of PacBio provides an introduction to PacBio HiFi sequence reads, which are both long (up to 25 kb currently) and accurate (>99%) at the individual single-molecule sequence read level andhave allowed for advances in de novo genome assemblies. Korlach reviews the characteristics of HiFi read data obtained with the Sequel II System, followed by examples of high-quality genome assemblies for human, plant and animal genomes including the different aspects of evaluating genome assemblies (contiguity, accuracy, completeness and allelic phasing) and illustrates their high quality by examples of resolving centromeres, telomeres, segmental duplications…
The sequence and assembly of human genomes using long-read sequencing technologies has revolutionized our understanding of structural variation and genome organization. We compared the accuracy, continuity, and gene annotation of genome assemblies generated from either high-fidelity (HiFi) or continuous long-read (CLR) datasets from the same complete hydatidiform mole human genome. We find that the HiFi sequence data assemble an additional 10% of duplicated regions and more accurately represent the structure of tandem repeats, as validated with orthogonal analyses. As a result, an additional 5 Mbp of pericentromeric sequences are recovered in the HiFi assembly, resulting in a 2.5-fold increase in…
The emergence of third generation sequencing (3GS; long-reads) is making closer the goal of chromosome-size fragments in de novo genome assemblies. This allows the exploration of new and broader questions on genome evolution for a number of non-model organisms. However, long-read technologies result in higher sequencing error rates and therefore impose an elevated cost of sufficient coverage to achieve high enough quality. In this context, hybrid assemblies, combining short-reads and long-reads provide an alternative efficient and cost-effective approach to generate de novo, chromosome-level genome assemblies. The array of available software programs for hybrid genome assembly, sequence correction and manipulation is…
Haplotype-resolved genome assemblies are important for understanding how combinations of variants impact phenotypes. These assemblies can be created in various ways, such as use of tissues that contain single-haplotype (haploid) genomes, or by co-sequencing of parental genomes, but these approaches can be impractical in many situations. We present FALCON-Phase, which integrates long-read sequencing data and ultra-long-range Hi-C chromatin interaction data of a diploid individual to create high-quality, phased diploid genome assemblies. The method was evaluated by application to three datasets, including human, cattle, and zebra finch, for which high-quality, fully haplotype resolved assemblies were available for benchmarking. Phasing algorithm accuracy…
Background New sequencing technologies have lowered financial barriers to whole genome sequencing, but resulting assemblies are often fragmented and far from textquoteleftfinishedtextquoteright. Updating multi-scaffold drafts to chromosome-level status can be achieved through experimental mapping or re-sequencing efforts. Avoiding the costs associated with such approaches, comparative genomic analysis of gene order conservation (synteny) to predict scaffold neighbours (adjacencies) offers a potentially useful complementary method for improving draft assemblies.Results We employed three gene synteny-based methods applied to 21 Anopheles mosquito assemblies to produce consensus sets of scaffold adjacencies. For subsets of the assemblies we integrated these with additional supporting data to confirm…
Brassica napus (AACC, 2n = 38) is an important oilseed crop grown worldwide. However, little is known about the population evolution of this species, the genomic difference between its major genetic groups, such as European and Asian rapeseed, and the impacts of historical large-scale introgression events on this young tetraploid. In this study, we reported the de novo assembly of the genome sequences of an Asian rapeseed (B. napus), Ningyou 7, and its four progenitors and compared these genomes with other available genomic data from diverse European and Asian cultivars. Our results showed that Asian rapeseed originally derived from European rapeseed but subsequently…
Background A high-quality reference genome is an essential tool for applied and basic research on arthropods. Long-read sequencing technologies may be used to generate more complete and contiguous genome assemblies than alternate technologies; however, long-read methods have historically had greater input DNA requirements and higher costs than next-generation sequencing, which are barriers to their use on many samples. Here, we present a 2.3 Gb de novo genome assembly of a field-collected adult female spotted lanternfly (Lycorma delicatula) using a single Pacific Biosciences SMRT Cell. The spotted lanternfly is an invasive species recently discovered in the northeastern United States that threatens…
The blood clam, Scapharca (Anadara) broughtonii, is an economically and ecologically important marine bivalve of the family Arcidae. Efforts to study their population genetics, breeding, cultivation, and stock enrichment have been somewhat hindered by the lack of a reference genome. Herein, we report the complete genome sequence of S. broughtonii, a first reference genome of the family Arcidae.A total of 75.79 Gb clean data were generated with the Pacific Biosciences and Oxford Nanopore platforms, which represented approximately 86× coverage of the S. broughtonii genome. De novo assembly of these long reads resulted in an 884.5-Mb genome, with a contig N50…
Yellowhorn (Xanthoceras sorbifolium) is a species of the Sapindaceae family native to China and is an oil tree that can withstand cold and drought conditions. A pseudomolecule-level genome assembly for this species will not only contribute to understanding the evolution of its genes and chromosomes but also bring yellowhorn breeding into the genomic era.Here, we generated 15 pseudomolecules of yellowhorn chromosomes, on which 97.04% of scaffolds were anchored, using the combined Illumina HiSeq, Pacific Biosciences Sequel, and Hi-C technologies. The length of the final yellowhorn genome assembly was 504.2 Mb with a contig N50 size of 1.04 Mb and a…
Lacerta viridis and Lacerta bilineata are sister species of European green lizards (eastern and western clades, respectively) that, until recently, were grouped together as the L. viridis complex. Genetic incompatibilities were observed between lacertid populations through crossing experiments, which led to the delineation of two separate species within the L. viridis complex. The population history of these sister species and processes driving divergence are unknown. We constructed the first high-quality de novo genome assemblies for both L. viridis and L. bilineata through Illumina and PacBio sequencing, with annotation support provided from transcriptome sequencing of several tissues. To estimate gene flow…
The adoption of single molecule real-time (SMRT) sequencing [1] is becoming widespread, not only in basic science, but also in more applied areas such as agricultural, environmental, and medical research. SMRT sequencing offers important advantages over current short-read DNA sequencing technologies, including exceptionally long read lengths (20 kb or more), unparalleled consensus accuracy, and the ability to sequence native, non-amplified, DNA molecules. These sequencing characteristics enable creation of highly accurate de novo genome assemblies, characterization of complex structural variation, direct characterization of nucleotide base modifications, full-length RNA isoform sequencing, phasing of genetic variants, low frequency mutation detection, and clonal evolution…