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June 1, 2021  |  

Genome sequencing of endosymbiotic bacterial Streptomyces sp. from Antartic lichen using Single Molecule Real-time Sequencing (SMRT) technology.

Along with the advent of next-generation sequencing (NGS) techniques, it has become possible to sequence a microbial genome very quickly with high coverage. Recently, PacificBioscience developed single molecule real-time sequencing (SMRT) technology, 3rd generation sequencing platform, which provide much longer (average read length: 1.5Kb) reads without PCR amplification. We did de novo sequencing of Streptomyces sp. using Illumina GAIIx, Roche 454 and PacBio RS system and compared the data. The endosymbiotic bacteria Streptomyces sp. PAMC 26508 was isolated from Antarctic lichen Psoroma sp. that grows attached rocks on Barton Peninsula, King George Island, Antarctica (62, 13’S, 58, 47’W). With 4 SMRT cells, we could get more than 15x coverage of corrected sequence data for de novo assembly. Comparing the performance of other sequencing platforms, PacBio platform could generate data on similar manner with general mid-level GC content organism. In conclusion, PacBio RS system, SMRT technology, shows better performance with high GC content organisms and is expected to be the new tool to improve the de novo sequencing and assembly.

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June 1, 2021  |  

SMRT Sequencing solutions for large genomes and transcriptomes.

Single Molecule, Real-Time (SMRT) Sequencing holds promise for addressing new frontiers in large genome complexities, such as long, highly repetitive, low-complexity regions and duplication events, and differentiating between transcript isoforms that are difficult to resolve with short-read technologies. We present solutions available for both reference genome improvement (>100 MB) and transcriptome research to best leverage long reads that have exceeded 20 Kb in length. Benefits for these applications are further realized with consistent use of size-selection of input sample using the BluePippin™ device from Sage Science. Highlights from our genome assembly projects using the latest P5-C3 chemistry on model organisms will be shared. Assembly contig N50 have exceeded 6 Mb and we observed longest contig exceeding 12.5 Mb with an average base quality of QV50. Additionally, the value of long, intact reads to provide a no-assembly approach to investigate transcript isoforms using our Iso-Seq Application will be presented.

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June 1, 2021  |  

Progress on the reassembly and annotation of the goat genome.

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.

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June 1, 2021  |  

The use of PacBio and Hi-C data in de novo assembly of the goat genome.

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.

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June 1, 2021  |  

A genome assembly of the domestic goat from 70x coverage of single molecule, real-time sequence.

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.

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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 and third-party solutions, including HGAP, MHAP, and ECTools. Resources and tools available on GitHub are reviewed, as well as datasets representing major model research organisms made publically available for community evaluation or interested developers.

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June 1, 2021  |  

Complete microbial genomes, epigenomes, and transcriptomes using long-read PacBio Sequencing.

For comprehensive metabolic reconstructions and a resulting understanding of the pathways leading to natural products, it is desirable to obtain complete information about the genetic blueprint of the organisms used. Traditional Sanger and next-generation, short-read sequencing technologies have shortcomings with respect to read lengths and DNA-sequence context bias, leading to fragmented and incomplete genome information. The development of long-read, single molecule, real-time (SMRT) DNA sequencing from Pacific Biosciences, with >10,000 bp average read lengths and a lack of sequence context bias, now allows for the generation of complete genomes in a fully automated workflow. In addition to the genome sequence, DNA methylation is characterized in the process of sequencing. PacBio® sequencing has also been applied to microbial transcriptomes. Long reads enable sequencing of full-length cDNAs allowing for identification of complete gene and operon sequences without the need for transcript assembly. We will highlight several examples where these capabilities have been leveraged in the areas of industrial microbiology, including biocommodities, biofuels, bioremediation, new bacteria with potential commercial applications, antibiotic discovery, and livestock/plant microbiome interactions.

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June 1, 2021  |  

Metagenomes of native and electrode-enriched microbial communities from the Soudan Iron Mine.

Despite apparent carbon limitation, anoxic deep subsurface brines at the Soudan Underground Iron Mine harbor active microbial communities. To characterize these assemblages, we performed shotgun metagenomics of native and enriched samples. Following enrichment on poised electrodes and long read sequencing, we recovered from the metagenome the closed, circular genome of a novel Desulfuromonas sp. with remarkable genomic features that were not fully resolved by short read assembly alone. This organism was essentially absent in unenriched Soudan communities, indicating that electrodes are highly selective for putative metal reducers. Native community metagenomes suggest that carbon cycling is driven by methyl-C1 metabolism, in particular methylotrophic methanogenesis. Our results highlight the promising potential for long reads in metagenomic surveys of low-diversity environments.

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June 1, 2021  |  

Introduction to SMRT informatics developers conference

2015 SMRT Informatics Developers Conference Presentation Slides: Kevin Corcoran of PacBio provided a brief review of community involvement in the development of analysis tools and showed a preview of upcoming sample preparation, chemistry and informatics improvements.

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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 with BioNano Genomics Irys optical maps and Lachesis clustering in order to provide a high quality reference assembly without the need for extensive filtering. Initial PacBio assemblies using P5C4 chemistry achieved contig N50’s of 4 Megabases and a BUSCO completion score of 84.0%, which is comparable to several finished model organism reference assemblies. We used BioNano Genomics’ Irys platform to generate 336 scaffolds from this data with a scaffold N50 of 24 megabases and total genome coverage of 98%. Lachesis interaction maps were used with a clustering algorithm to associate Irys scaffolds into the expected 30 chromosome physical maps. Comparisons of the initial hybrid scaffolds generated from the long read contigs and optical map information to a previously generated RH map revealed that the entirety of the Goat autosome 20 physical map was contained within one scaffold. Additionally, the BioNano scaffolding resolved several difficult regions that contained genes related to innate immunity which were problem regions in previous reference genome assemblies.

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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 the MinHash Alignment Process (MHAP) with the Celera Assembler (CA) to produce an assembly composed of 3110 contigs with a contig N50 size of 4.7 Mb. This assembly was scaffolded with BioNano genome maps derived from a single IrysChip into 333 scaffolds with an N50 of 23.1 Mb including the complete scaffolding of chromosome 20. Finally, cis-chromosome associations were determined by Hi-C, yielding complete reconstruction of all autosomes into single scaffolds with a final N50 of 91.7 Mb. We hope to demonstrate that our methods are not only cost effective, but improve our ability to annotate challenging genomic regions such as highly repetitive immune gene clusters.

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April 21, 2020  |  

A High-Quality Grapevine Downy Mildew Genome Assembly Reveals Rapidly Evolving and Lineage-Specific Putative Host Adaptation Genes.

Downy mildews are obligate biotrophic oomycete pathogens that cause devastating plant diseases on economically important crops. Plasmopara viticola is the causal agent of grapevine downy mildew, a major disease in vineyards worldwide. We sequenced the genome of Pl. viticola with PacBio long reads and obtained a new 92.94?Mb assembly with high contiguity (359 scaffolds for a N50 of 706.5?kb) due to a better resolution of repeat regions. This assembly presented a high level of gene completeness, recovering 1,592 genes encoding secreted proteins involved in plant-pathogen interactions. Plasmopara viticola had a two-speed genome architecture, with secreted protein-encoding genes preferentially located in gene-sparse, repeat-rich regions and evolving rapidly, as indicated by pairwise dN/dS values. We also used short reads to assemble the genome of Plasmopara muralis, a closely related species infecting grape ivy (Parthenocissus tricuspidata). The lineage-specific proteins identified by comparative genomics analysis included a large proportion of RxLR cytoplasmic effectors and, more generally, genes with high dN/dS values. We identified 270 candidate genes under positive selection, including several genes encoding transporters and components of the RNA machinery potentially involved in host specialization. Finally, the Pl. viticola genome assembly generated here will allow the development of robust population genomics approaches for investigating the mechanisms involved in adaptation to biotic and abiotic selective pressures in this species. © The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Molecular Biology and Evolution.

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April 21, 2020  |  

Comprehensive evaluation of non-hybrid genome assembly tools for third-generation PacBio long-read sequence data.

Long reads obtained from third-generation sequencing platforms can help overcome the long-standing challenge of the de novo assembly of sequences for the genomic analysis of non-model eukaryotic organisms. Numerous long-read-aided de novo assemblies have been published recently, which exhibited superior quality of the assembled genomes in comparison with those achieved using earlier second-generation sequencing technologies. Evaluating assemblies is important in guiding the appropriate choice for specific research needs. In this study, we evaluated 10 long-read assemblers using a variety of metrics on Pacific Biosciences (PacBio) data sets from different taxonomic categories with considerable differences in genome size. The results allowed us to narrow down the list to a few assemblers that can be effectively applied to eukaryotic assembly projects. Moreover, we highlight how best to use limited genomic resources for effectively evaluating the genome assemblies of non-model organisms. © The Author 2017. Published by Oxford University Press.

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April 21, 2020  |  

Characterizing the major structural variant alleles of the human genome.

In order to provide a comprehensive resource for human structural variants (SVs), we generated long-read sequence data and analyzed SVs for fifteen human genomes. We sequence resolved 99,604 insertions, deletions, and inversions including 2,238 (1.6 Mbp) that are shared among all discovery genomes with an additional 13,053 (6.9 Mbp) present in the majority, indicating minor alleles or errors in the reference. Genotyping in 440 additional genomes confirms the most common SVs in unique euchromatin are now sequence resolved. We report a ninefold SV bias toward the last 5 Mbp of human chromosomes with nearly 55% of all VNTRs (variable number of tandem repeats) mapping to this portion of the genome. We identify SVs affecting coding and noncoding regulatory loci improving annotation and interpretation of functional variation. These data provide the framework to construct a canonical human reference and a resource for developing advanced representations capable of capturing allelic diversity. Copyright © 2018 Elsevier Inc. All rights reserved.

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