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Friday, February 5, 2021

AGBT Presentation: Feed the World – Developing genomic resources for insects as food

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.

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Friday, February 5, 2021

Video Poster: Full-length RNA sequencing of Alzheimer brain sample using long reads reveals complex alternative splicing patterns

Long-read mRNA sequencing such as PacBio’s Iso-Seq method offer high-throughput transcriptome profiling that circumvents the transcript assembly problem by sequencing full-length cDNA. The Iso-Seq method has emerged as the most reliable technology for fully characterizing isoforms and, in turn, help shed light on underlying disease mechanisms. Here, we have utilized the Iso-Seq method to sequence an Alzheimer’s disease whole brain?sample. This is a devastating neurodegenerative disease that affects ~44 million people worldwide, making it the most common form of dementia. Studies looking into disease mechanism have shown that changes in gene expression due to alternative splicing likely contribute to the…

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Friday, February 5, 2021

ASHG CoLab: PacBio HiFi reads for comprehensive characterization of genomes and single-cell isoform expression

In this ASHG 2020 CoLab presentation hear Principal Scientists, Aaron Wenger and Elizabeth Tseng share how highly accurate long reads (HiFi reads) provide comprehensive variant detection for both genomes and transcriptomes. Aaron Wenger describes how new improvements in protocols and analysis methods have increased scalability and accuracy of variant calling. As demonstrated in the precisionFDA Truth Challenge V2, HiFi reads (>99% accurate, 15 kb – 20 kb) now outperform short reads for single nucleotide and structural variant calling and match for small indels. This includes calling >30,000 small variants and >10,000 structural variants missed by short reads, many in medically…

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Tuesday, April 21, 2020

Long-read sequencing for rare human genetic diseases.

During the past decade, the search for pathogenic mutations in rare human genetic diseases has involved huge efforts to sequence coding regions, or the entire genome, using massively parallel short-read sequencers. However, the approximate current diagnostic rate is

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Tuesday, April 21, 2020

Characterization of Reference Materials for Genetic Testing of CYP2D6 Alleles: A GeT-RM Collaborative Project.

Pharmacogenetic testing increasingly is available from clinical and research laboratories. However, only a limited number of quality control and other reference materials currently are available for the complex rearrangements and rare variants that occur in the CYP2D6 gene. To address this need, the Division of Laboratory Systems, CDC-based Genetic Testing Reference Material Coordination Program, in collaboration with members of the pharmacogenetic testing and research communities and the Coriell Cell Repositories (Camden, NJ), has characterized 179 DNA samples derived from Coriell cell lines. Testing included the recharacterization of 137 genomic DNAs that were genotyped in previous Genetic Testing Reference Material Coordination…

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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

RNA sequencing: the teenage years.

Over the past decade, RNA sequencing (RNA-seq) has become an indispensable tool for transcriptome-wide analysis of differential gene expression and differential splicing of mRNAs. However, as next-generation sequencing technologies have developed, so too has RNA-seq. Now, RNA-seq methods are available for studying many different aspects of RNA biology, including single-cell gene expression, translation (the translatome) and RNA structure (the structurome). Exciting new applications are being explored, such as spatial transcriptomics (spatialomics). Together with new long-read and direct RNA-seq technologies and better computational tools for data analysis, innovations in RNA-seq are contributing to a fuller understanding of RNA biology, from questions…

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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

Towards PacBio-based pan-eukaryote metabarcoding using full-length ITS sequences.

Development of high-throughput sequencing techniques have greatly benefited our understanding about microbial ecology; yet the methods producing short reads suffer from species-level resolution and uncertainty of identification. Here we optimize PacBio-based metabarcoding protocols covering the Internal Transcribed Spacer (ITS region) and partial Small Subunit (SSU) of the rRNA gene for species-level identification of all eukaryotes, with a specific focus on Fungi (including Glomeromycota) and Stramenopila (particularly Oomycota). Based on tests on composite soil samples and mock communities, we propose best suitable degenerate primers, ITS9munngs + ITS4ngsUni for eukaryotes and selected groups therein and discuss pros and cons of long read-based…

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Tuesday, April 21, 2020

deSALT: fast and accurate long transcriptomic read alignment with de Bruijn graph-based index

Long-read RNA sequencing (RNA-seq) is promising to transcriptomics studies, however, the alignment of the reads is still a fundamental but non-trivial task due to the sequencing errors and complicated gene structures. We propose deSALT, a tailored two-pass long RNA-seq read alignment approach, which constructs graph-based alignment skeletons to sensitively infer exons, and use them to generate spliced reference sequence to produce refined alignments. deSALT addresses several difficult issues, such as small exons, serious sequencing errors and consensus spliced alignment. Benchmarks demonstrate that this approach has a better ability to produce high-quality full-length alignments, which has enormous potentials to transcriptomics studies.

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Tuesday, April 21, 2020

Full-length mRNA sequencing and gene expression profiling reveal broad involvement of natural antisense transcript gene pairs in pepper development and response to stresses.

Pepper is an important vegetable with great economic value and unique biological features. In the past few years, significant development has been made towards understanding the huge complex pepper genome; however, pepper functional genomics has not been well studied. To better understand the pepper gene structure and pepper gene regulation, we conducted full-length mRNA sequencing by PacBio sequencing and obtained 57862 high-quality full-length mRNA sequences derived from 18362 previously annotated and 5769 newly detected genes. New gene models were built that combined the full-length mRNA sequences and corrected approximately 500 fragmented gene models from previous annotations. Based on the full-length…

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Tuesday, April 21, 2020

Defining transgene insertion sites and off-target effects of homology-based gene silencing informs the use of functional genomics tools in Phytophthora infestans.

DNA transformation and homology-based transcriptional silencing are frequently used to assess gene function in Phytophthora. Since unplanned side-effects of these tools are not well-characterized, we used P. infestans to study plasmid integration sites and whether knockdowns caused by homology-dependent silencing spreads to other genes. Insertions occurred both in gene-dense and gene-sparse regions but disproportionately near the 5′ ends of genes, which disrupted native coding sequences. Microhomology at the recombination site between plasmid and chromosome was common. Studies of transformants silenced for twelve different gene targets indicated that neighbors within 500-nt were often co-silenced, regardless of whether hairpin or sense constructs…

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Tuesday, April 21, 2020

Lateral transfers of large DNA fragments spread functional genes among grasses.

A fundamental tenet of multicellular eukaryotic evolution is that vertical inheritance is paramount, with natural selection acting on genetic variants transferred from parents to offspring. This lineal process means that an organism’s adaptive potential can be restricted by its evolutionary history, the amount of standing genetic variation, and its mutation rate. Lateral gene transfer (LGT) theoretically provides a mechanism to bypass many of these limitations, but the evolutionary importance and frequency of this process in multicellular eukaryotes, such as plants, remains debated. We address this issue by assembling a chromosome-level genome for the grass Alloteropsis semialata, a species surmised to…

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Tuesday, April 21, 2020

The landscape of SNCA transcripts across synucleinopathies: New insights from long reads sequencing analysis

Dysregulation of alpha-synuclein expression has been implicated in the pathogenesis of synucleinopathies, in particular Parkinsontextquoterights Disease (PD) and Dementia with Lewy bodies (DLB). Previous studies have shown that the alternatively spliced isoforms of the SNCA gene are differentially expressed in different parts of the brain for PD and DLB patients. Similarly, SNCA isoforms with skipped exons can have a functional impact on the protein domains. The large intronic region of the SNCA gene was also shown to harbor structural variants that affect transcriptional levels. Here we apply the first study of using long read sequencing with targeted capture of both…

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Tuesday, April 21, 2020

Optimized Cas9 expression systems for highly efficient Arabidopsis genome editing facilitate isolation of complex alleles in a single generation.

Genetic resources for the model plant Arabidopsis comprise mutant lines defective in almost any single gene in reference accession Columbia. However, gene redundancy and/or close linkage often render it extremely laborious or even impossible to isolate a desired line lacking a specific function or set of genes from segregating populations. Therefore, we here evaluated strategies and efficiencies for the inactivation of multiple genes by Cas9-based nucleases and multiplexing. In first attempts, we succeeded in isolating a mutant line carrying a 70 kb deletion, which occurred at a frequency of ~?1.6% in the T2 generation, through PCR-based screening of numerous individuals. However,…

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