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Tuesday, June 1, 2021

Genome sequencing of microbial genomes using Single Molecule Real-time sequencing (SMRT) technology.

In the last year, high-throughput sequencing technologies have progressed from proof-of-concept to production quality. Although each technology is able to produce vast quantities of sequence information, in every case the underlying chemistry limits reads to very short lengths. We present a examining de novo assembly comparison with bacterial genome assembly varying genome size (from 3.1Mb to 7.6Mb) and different G+C contents (from 43% to 71%), respectively. We analyzed Solexa reads, 454 reads and Pacbio RS reads from Streptomyces sp. (Genome size, 7.6 Mb; G+C content, 71%), Psychrobacter sp. (Genome size, 3.5 Mb; G+C content, 43%), Salinibacterium sp. (Genome size, 3.1…

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Tuesday, June 1, 2021

Advances in sequence consensus and clustering algorithms for effective de novo assembly and haplotyping applications.

One of the major applications of DNA sequencing technology is to bring together information that is distant in sequence space so that understanding genome structure and function becomes easier on a large scale. The Single Molecule Real Time (SMRT) Sequencing platform provides direct sequencing data that can span several thousand bases to tens of thousands of bases in a high-throughput fashion. In contrast to solving genomic puzzles by patching together smaller piece of information, long sequence reads can decrease potential computation complexity by reducing combinatorial factors significantly. We demonstrate algorithmic approaches to construct accurate consensus when the differences between reads…

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Tuesday, June 1, 2021

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens.

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single-nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non- pathogenic to pathogenic…

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Tuesday, June 1, 2021

Automated, non-hybrid de novo genome assemblies and epigenomes of bacterial pathogens

Understanding the genetic basis of infectious diseases is critical to enacting effective treatments, and several large-scale sequencing initiatives are underway to collect this information. Sequencing bacterial samples is typically performed by mapping sequence reads against genomes of known reference strains. While such resequencing informs on the spectrum of single nucleotide differences relative to the chosen reference, it can miss numerous other forms of variation known to influence pathogenicity: structural variations (duplications, inversions), acquisition of mobile elements (phages, plasmids), homonucleotide length variation causing phase variation, and epigenetic marks (methylation, phosphorothioation) that influence gene expression to switch bacteria from non-pathogenic to pathogenic…

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Tuesday, June 1, 2021

Old school/new school genome sequencing: One step backward — a quantum leap forward.

As the costs for genome sequencing have decreased the number of “genome” sequences have increased at a rapid pace. Unfortunately, the quality and completeness of these so–called “genome” sequences have suffered enormously. We prefer to call such genome assemblies as “gene assembly space” (GAS). We believe it is important to distinguish GAS assemblies from reference genome assemblies (RGAs) as all subsequent research that depends on accurate genome assemblies can be highly compromised if the only assembly available is a GAS assembly.

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Tuesday, June 1, 2021

Draft genome of horseweed illuminates expansion of gene families that might endow herbicide resistance.

Conyza canadensis (horseweed), a member of the Compositae (Asteraceae) family, was the first broadleaf weed to evolve resistance to glyphosate. Horseweed, one of the most problematic weeds in the world, is a true diploid (2n=2X=18) with the smallest genome of any known agricultural weed (335 Mb). Thus, it is an appropriate candidate to help us understand the genetic and genomic basis of weediness. We undertook a draft de novo genome assembly of horseweed by combining data from multiple sequencing platforms (454 GS-FLX, Illumina HiSeq 2000 and PacBio RS) using various libraries with different insertion sizes (~350 bp, ~600 bp, ~3…

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Tuesday, June 1, 2021

Genome analysis of a bacterium that causes lameness.

Lameness is a significant problem resulting in millions of dollars in lost revenue annually. In commercial broilers, the most common cause of lameness is bacterial chondronecrosis with osteomyelitis (BCO). We are using a wire flooring model to induce lameness attributable to BCO. We used 16S ribosomal DNA sequencing to determine that Staphylococcus spp. were the main species associated with BCO. Staphylococcus agnetis, which previously had not been isolated from poultry, was the principal species isolated from the majority of the bone lesion samples. Administering S. agnetis in the drinking water to broilers reared on wire flooring increased the incidence of…

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Tuesday, June 1, 2021

Resources for advanced bioinformaticians working in plant and animal genomes with SMRT Sequencing.

Significant advances in bioinformatics tool development have been made to more efficiently leverage and deliver high-quality genome assemblies with PacBio long-read data. Current data throughput of SMRT Sequencing delivers average read lengths ranging from 10-15 kb with the longest reads exceeding 40 kb. This has resulted in consistent demonstration of a minimum 10-fold improvement in genome assemblies with contig N50 in the megabase range compared to assemblies generated using only short- read technologies. This poster highlights recent advances and resources available for advanced bioinformaticians and developers interested in the current state-of-the-art large genome solutions available as open-source code from PacBio…

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Tuesday, June 1, 2021

The resurgence of reference quality genome sequence.

Since the advent of Next-Generation Sequencing (NGS), the cost of de novo genome sequencing and assembly have dropped precipitately, which has spurred interest in genome sequencing overall. Unfortunately the contiguity of the NGS assembled sequences, as well as the accuracy of these assemblies have suffered. Additionally, most NGS de novo assemblies leave large portions of genomes unresolved, and repetitive regions are often collapsed. When compared to the reference quality genome sequences produced before the NGS era, the new sequences are highly fragmented and often prove to be difficult to properly annotate. In some cases the contiguous portions are smaller than…

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Tuesday, June 1, 2021

Genome in a Bottle: You’ve sequenced. How well did you do?

Purpose: Clinical laboratories, research laboratories and technology developers all need DNA samples with reliably known genotypes in order to help validate and improve their methods. The Genome in a Bottle Consortium (genomeinabottle.org) has been developing Reference Materials with high-accuracy whole genome sequences to support these efforts.Methodology: Our pilot reference material is based on Coriell sample NA12878 and was released in May 2015 as NIST RM 8398 (tinyurl.com/giabpilot). To minimize bias and improve accuracy, 11 whole-genome and 3 exome data sets produced using 5 different technologies were integrated using a systematic arbitration method [1]. The Genome in a Bottle Analysis Group…

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Tuesday, June 1, 2021

Genome and transcriptome of the refeneration-competent flatworm, Macrostomum lignano

The free-living flatworm, Macrostomum lignano, much like its better known planarian relative, Schmidtea mediterranea, has an impressive regenerative capacity. Following injury, this species has the ability to regenerate almost an entirely new organism. This is attributable to the presence of an abundant somatic stem cell population, the neoblasts. These cells are also essential for the ongoing maintenance of most tissues, as their loss leads to irreversible degeneration of the animal. This set of unique properties makes a subset of flatworms attractive organisms for studying the evolution of pathways involved in tissue self-renewal, cell fate specification, and regeneration. The use of…

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Tuesday, June 1, 2021

Improving the goat long-read assembly with optical mapping and Hi-C scaffolding

Reference genome assemblies provide important context in genetics by standardizing the order of genes and providing a universal set of coordinates for individual nucleotides. Often due to the high complexity of genic regions and higher copy number of genes involved in immune function, immunity-related genes are often misassembled in current reference assemblies. This problem is particularly ubiquitous in the reference genomes of non-model organisms as they often do not receive the years of curation necessary to resolve annotation and assembly errors. In this study, we reassemble a reference genome of the goat (Capra hircus) using modern PacBio technology in tandem…

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Tuesday, June 1, 2021

Progress Toward a Low Budget Reference Grade Genome Assembly

Reference quality de novo genome assemblies were once solely the domain of large, well-funded genome projects. While next-generation short read technology removed some of the cost barriers, accurate chromosome-scale assembly remains a real challenge. Here we present efforts to de novo assemble the goat (Capra hircus) genome. Through the combination of single-molecule technologies from Pacific Biosciences (sequencing) and BioNano Genomics (optical mapping) coupled with high-throughput chromosome conformation capture sequencing (Hi-C), an inbred San Clemente goat genome has been sequenced and assembled to a high degree of completeness at a relatively modest cost. Starting with 38 million PacBio reads, we integrated…

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Tuesday, June 1, 2021

Phased diploid genome assembly with single-molecule real-time sequencing

While genome assembly projects have been successful in many haploid and inbred species, the assembly of non-inbred or rearranged heterozygous genomes remains a major challenge. To address this challenge, we introduce the open-source FALCON and FALCON-Unzip algorithms (https://github.com/PacificBiosciences/FALCON/) to assemble long-read sequencing data into highly accurate, contiguous, and correctly phased diploid genomes. We generate new reference sequences for heterozygous samples including an F1 hybrid of Arabidopsis thaliana, the widely cultivated Vitis vinifera cv. Cabernet Sauvignon, and the coral fungus Clavicorona pyxidata, samples that have challenged short-read assembly approaches. The FALCON-based assemblies are substantially more contiguous and complete than alternate short-…

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