In this talk, Dr. Stephanie Tome describes using PacBio Single Molecule, Real-Time (SMRT) Sequencing to precisely measure large CTG repeat size and identify sequence interruptions of expanded allele to understand…
In this talk, Dr. Zachary McEachin describes how No-Amp targeted sequencing enables sequencing analysis of multiple (at least 15) Ataxia-related repeat expansion loci in one assay, presenting example sequencing results…
AGBT 2013 Presentation Slides: Cold Spring Harbor Laboratory’s Michael Schatz presented strategies for de novo assembly of crop genomes with PacBio technolgy.
PacBio 2013 User Group Meeting Presentation Slides: Lisbeth Guethlein from Stanford University School of Medicine looked at highly repetitive and variable immune regions of the orangutan genome. Guethlein reported that “PacBio managed to accomplish in a week what I have been working on for a couple years” (with Sanger sequencing), and the results were concordant. “Long story short, I was a happy customer.”
PacBio 2013 User Group Meeting Presentation Slides: Lance Hepler from UC San Diego’s Center for AIDS Research used the PacBio RS to study intra-host diversity in HIV-1. He compared PacBio’s performance to that of 454® sequencer, the platform he and his team previously used. Hepler noted that in general, there was strong agreement between the platforms; where results differed, he said that PacBio data had significantly better reproducibility and accuracy. “PacBio does not suffer from local coverage loss post-processing, whereas 454 has homopolymer problems,” he noted. Hepler said they are moving away from using 454 in favor of the PacBio system.
SFAF 2014 Presentation Slides: James Gurtowski of Cold Spring Harbor Laboratory (CSHL) shared assembly results for a variety of eukaryotic genomes, including yeast, arabidopsis, and rice.
PacBio 2014 User Group Meeting Presentation Slides: Anne Deslattes Mays of Georgetown University discussed how PacBio provided the necessary full-length isoform information to allow characterization of isoform distribution by sub-cell population.
PacBio 2014 User Group Meeting Presentation Slides: Alisha Holloway of the Gladstone Institutes presented on the use of isoform sequencing (Iso-Seq) to improve the annotation of the chicken genome as a model reference for cardiovascular research.
Generating de novo reference genome assemblies for non-model organisms is a laborious task that often requires a large amount of data from several sequencing platforms and cytogenetic surveys. By using PacBio sequence data and new library creation techniques, we present a de novo, high quality reference assembly for the goat (Capra hircus) that demonstrates a primarily sequencing-based approach to efficiently create new reference assemblies for Eukaryotic species. This goat reference genome was created using 38 million PacBio P5-C3 reads generated from a San Clemente goat using the Celera Assembler PBcR pipeline with PacBio read self-correction. In order to generate the assembly, corrected and filtered reads were pre-assembled into a consensus model using PBDAGCON, and subsequently assembled using the Celera Assembly version 8.2. We generated 5,902 contigs using this method with a contig N50 size of 2.56 megabases. In order to generate chromosome-sized scaffolds, we used the LACHESIS scaffolding method to identify cis-chromosome Hi-C interactions in order to link contigs together. We then compared our new assembly to the existing goat reference assembly to identify large-scale discrepancies. In our comparison, we identified 247 disagreements between the two assemblies consisting of 123 inversions and 124 chromosome-contig relocations. The high quality of this data illustrates how this methodology can be used to efficiently generate new reference genome assemblies without the use of expensive fluorescent cytometry or large quantities of data from multiple sequencing platforms.
The goat (Capra hircus) remains an important livestock species due to the species’ ability to forage and provide milk, meat and wool in arid environments. The current goat reference assembly and annotation borrows heavily from other loosely related livestock species, such as cattle, and may not reflect the unique structural and functional characteristics of the species. We present preliminary data from a new de novo reference assembly for goat that primarily utilizes 38 million PacBio P5-C3 reads generated from an inbred San Clemente goat. This assembly consists of only 5,902 contigs with a contig N50 size of 2.56 megabases which were grouped into scaffolds using cis-chromosome associations generated by the analysis of Hi-C sequence reads. To provide accurate functional genetic annotation, we utilized existing RNA-seq data and generated new data consisting of over 784 million reads from a combination of 27 different developmental timepoints/tissues. This dataset provides a tangible improvement over existing goat genomics resources by correcting over 247 misassemblies in the current goat reference genome and by annotating predicted gene models with actual expressed transcript data. Our goal is to provide a high quality resource to researchers to enable future genomic selection and functional prediction within the field of goat genomics.
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 kb and ~10 kb) of a Tennessee-accessed, glyphosate-resistant horseweed biotype. From 116.3 Gb (~350× coverage) of data, the genome was assembled into 13,966 scaffolds with N50 =33,561 bp. The assembly covered 92.3% of the genome, including the complete chloroplast genome (~153 kb) and a nearly-complete mitochondrial genome (~450 kb in 120 scaffolds). The nuclear genome is comprised of 44,592 protein-coding genes. Genome re-sequencing of seven additional horseweed biotypes was performed. These sequence data were assembled and used to analyze genome variation. Simple sequence repeat and single nucleotide polymorphisms were surveyed. Genomic patterns were detected that associated with glyphosate-resistant or –susceptible biotypes. The draft genome will be useful to better understand weediness, the evolution of herbicide resistance, and to devise new management strategies. The genome will also be useful as another reference genome in the Compositae. To our knowledge, this paper represents the first published draft genome of an agricultural weed.
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.
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 BCO three-fold when compared with broilers drinking tap water (P = 0.001). We found that the minimum effective dose of Staphylococcus agnetis to induce BCO in broilers grown on wire flooring experiment is 105 cfu/ml. We used PacBio and Illumina sequencing to assemble a 2.4 Mbp contig representing the genome and a 34 kbp contig for the largest plasmid of S. agnetis. Annotation of this genome is underway through comparative genomics with other Staphylococcus genomes, and identification of virulence factors. Our goal is to elucidate genetic diversity, toxins, and pathogenicity determinants, for this poorly characterized species. Isolating pathogenic bacterial species, defining their likely route of transmission to broilers, and genomic analyses will contribute substantially to the development of measures for mitigating BCO losses in poultry.
Goat is an important source of milk, meat, and fiber, especially in developing countries. An advantage of goats as livestock is the low maintenance requirements and high adaptability compared to other milk producers. The global population of domestic goats exceeds 800 million. In Africa, goat production is characterized by low productivity levels, and attempts to introduce more productive breeds have met with poor success due in part to nutritional constraints. It has been suggested that incorporation of selective breeding within the herds adapted for survival could represent one approach to improving food security across Africa. A recently produced genome assembly of a Chinese Yunnan breed goat, based on 192 Gb of short reads across a range of insert sizes from 180 bp to 20 kb, reported a contig N50 of 18.7 kb. The scaffold N50 was improved from 2.2 Mb to 3.1 Mb by addition of fosmid end sequence, with an estimated 140 million Ns in gaps and 91% coverage. The assembly has proven somewhat problematic for pursuing genome-wide association analysis with SNP arrays, apparently due in part to errors in ordering of markers using the draft genome. In order to provide a higher quality assembly, we sequenced a highly inbred, San Clemente breed goat genome using 458 SMRT cells on the Pacific Biosciences platform. These cells generated 193.5 Gbases of sequence after processing into subreads, with mean 5110 bases and max subread length of 40.5 kb. This sequence data generated an assembly using the recently reported MHAP error correction approach and Celera Assembler v8.2. The contig N50 was 2.5 Mb, with the largest contig spanning 19.5 Mb. Additional characteristics of the assembly will be presented.
Drought is responsible for much of the global losses in crop yields and understanding how plants naturally cope with drought stress is essential for breeding and engineering crops for the changing climate. Resurrection plants desiccate to complete dryness during times of drought, then “come back to life” once water is available making them an excellent model for studying drought tolerance. Understanding the molecular networks governing how resurrection plants handle desiccation will provide targets for crop engineering. Oropetium thomaeum (Oro) is a resurrection plant that also has the smallest known grass genome at 250 Mb compared to Brachypodium distachyon (300 Mb) and rice (350 Mb). Plant genomes, especially grasses, have complex repeat structures such as telomeres, centromeres, and ribosomal gene cassettes, and high heterozygosity, which makes them difficult to assembly using short read next generation sequencing technologies. Ultra-long PacBio reads using the new P6C4 chemistry and the latest 15kb Blue Pippin size-selection protocol to generate 20kb insert libraries that yielded an average read length of 12kb providing ~72X coverage, and 10X coverage with reads over 20kb. The HGAP assembly covers 98% of the genome with a contig N50 of 2.4 Mb, which makes it one of the highest quality and most complete plant genomes assembled to date. Oro has a compact genome structure compared to other grasses with only 16% repeat sequences but has very good collinearity with other grasses. Understanding the genomic mechanisms of extreme desiccation tolerance in resurrection plants like Oro will provide insights for engineering and intelligent breeding of improved food, fuel, and fiber crops.
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