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Friday, February 5, 2021

User Group Meeting: Development, validation, and applications of a high-fidelity, species-specific, eubacterial pan-domain microbiome assay on the PacBio System

In this PacBio User Group Meeting presentation, Garth Ehrlich of Drexel University College of Medicine shares his work on developing a microbiome assay that uses SMRT Sequencing to provide high-quality coverage of the 16S bacterial rRNA for species identification. The microbiome analysis pipeline, MCSMRT, takes advantage of PacBio circular consensus sequencing (CCS) technology and second-generation pathway analysis system for generating extremely high-fidelity sequences that provide the user with ultra-high-confidence species-level microbiome data.

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Friday, February 5, 2021

User Group Meeting: Application of genome assembly in Bovinae species

In this PacBio User Group Meeting presentation, Tim Smith of the USDA’s Agricultural Research Service describes efforts to generate reference-grade genome assemblies for various bovine species and analyze them to understand factors such as how selective breeding has affected certain breeds. Genome assemblies he presents span cattle, water buffalo, and gaur. Smith shows data for each assembly, noting that as data production shifted to the Sequel System, long-read PacBio data became even better at producing highly contiguous assemblies.

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Friday, February 5, 2021

User Group Meeting: The trials and tribulations of high quality human genome assembly

In this PacBio User Group Meeting presentation, Tina Graves-Lindsay of the McDonnell Genome Institute and the Genome Reference Consortium speaks about the importance of phasing human reference genomes. Her team is now working on its fifteenth human genome assembly — part of a major effort to improve genomic representation of ethnic diversity — with a pipeline that generates 60-fold PacBio coverage for a de novo assembly, followed by scaffolding with other technologies. They are also using FALCON-Unzip to separate haplotypes, leading to reference-grade diploid assemblies. This approach has already helped resolve errors seen in other genomes and even the gold-standard…

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Friday, February 5, 2021

AGBT Presentation: Generating high quality human reference assemblies with PacBio sequencing

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.

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Tuesday, April 21, 2020

Improved assembly and variant detection of a haploid human genome using single-molecule, high-fidelity long reads.

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…

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Tuesday, April 21, 2020

Chlorella vulgaris genome assembly and annotation reveals the molecular basis for metabolic acclimation to high light conditions.

Chlorella vulgaris is a fast-growing fresh-water microalga cultivated at the industrial scale for applications ranging from food to biofuel production. To advance our understanding of its biology and to establish genetics tools for biotechnological manipulation, we sequenced the nuclear and organelle genomes of Chlorella vulgaris 211/11P by combining next generation sequencing and optical mapping of isolated DNA molecules. This hybrid approach allowed to assemble the nuclear genome in 14 pseudo-molecules with an N50 of 2.8 Mb and 98.9% of scaffolded genome. The integration of RNA-seq data obtained at two different irradiances of growth (high light-HL versus low light -LL) enabled…

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Tuesday, April 21, 2020

Chromosome-length haplotigs for yak and cattle from trio binning assembly of an F1 hybrid

Background Assemblies of diploid genomes are generally unphased, pseudo-haploid representations that do not correctly reconstruct the two parental haplotypes present in the individual sequenced. Instead, the assembly alternates between parental haplotypes and may contain duplications in regions where the parental haplotypes are sufficiently different. Trio binning is an approach to genome assembly that uses short reads from both parents to classify long reads from the offspring according to maternal or paternal haplotype origin, and is thus helped rather than impeded by heterozygosity. Using this approach, it is possible to derive two assemblies from an individual, accurately representing both parental contributions…

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Tuesday, April 21, 2020

A robust benchmark for germline structural variant detection

New technologies and analysis methods are enabling genomic structural variants (SVs) to be detected with ever-increasing accuracy, resolution, and comprehensiveness. Translating these methods to routine research and clinical practice requires robust benchmark sets. We developed the first benchmark set for identification of both false negative and false positive germline SVs, which complements recent efforts emphasizing increasingly comprehensive characterization of SVs. To create this benchmark for a broadly consented son in a Personal Genome Project trio with broadly available cells and DNA, the Genome in a Bottle (GIAB) Consortium integrated 19 sequence-resolved variant calling methods, both alignment- and de novo assembly-based,…

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Tuesday, April 21, 2020

Genome assembly provides insights into the genome evolution and flowering regulation of orchardgrass.

Orchardgrass (Dactylis glomerata L.) is an important forage grass for cultivating livestock worldwide. Here, we report an ~1.84-Gb chromosome-scale diploid genome assembly of orchardgrass, with a contig N50 of 0.93 Mb, a scaffold N50 of 6.08 Mb and a super-scaffold N50 of 252.52 Mb, which is the first chromosome-scale assembled genome of a cool-season forage grass. The genome includes 40 088 protein-coding genes, and 69% of the assembled sequences are transposable elements, with long terminal repeats (LTRs) being the most abundant. The LTRretrotransposons may have been activated and expanded in the grass genome in response to environmental changes during the Pleistocene between 0 and…

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Tuesday, April 21, 2020

Insect genomes: progress and challenges.

In the wake of constant improvements in sequencing technologies, numerous insect genomes have been sequenced. Currently, 1219 insect genome-sequencing projects have been registered with the National Center for Biotechnology Information, including 401 that have genome assemblies and 155 with an official gene set of annotated protein-coding genes. Comparative genomics analysis showed that the expansion or contraction of gene families was associated with well-studied physiological traits such as immune system, metabolic detoxification, parasitism and polyphagy in insects. Here, we summarize the progress of insect genome sequencing, with an emphasis on how this impacts research on pest control. We begin with a…

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Tuesday, April 21, 2020

Extended haplotype phasing of de novo genome assemblies with FALCON-Phase

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…

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