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Tuesday, June 1, 2021

Impact of DNA quality on PacBio RS II read lengths.

Maximizing the read length of next generation sequencing (NGS) facilitates de novo genome assembly. Currently, the PacBio RS II system leads the industry with respect to maximum possible NGS read lengths. Amplicon Express specializes in preparation of high molecular weight, NGS-grade genomic DNA for a variety of applications, including next generation sequencing. This study was performed to evaluate the effects of gDNA quality on PacBio RS II read length.

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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

Single Molecule, Real-Time sequencing of full-length cDNA transcripts uncovers novel alternatively spliced isoforms.

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties such as structure, function, or subcellular localization. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be underscored. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey…

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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

Targeted SMRT Sequencing and phasing using Roche NimbleGen’s SeqCap EZ enrichment

As a cost-effective alternative to whole genome human sequencing, targeted sequencing of specific regions, such as exomes or panels of relevant genes, has become increasingly common. These methods typically include direct PCR amplification of the genomic DNA of interest, or the capture of these targets via probe-based hybridization. Commonly, these approaches are designed to amplify or capture exonic regions and thereby result in amplicons or fragments that are a few hundred base pairs in length, a length that is well-addressed with short-read sequencing technologies. These approaches typically provide very good coverage and can identify SNPs in the targeted region, but…

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Tuesday, June 1, 2021

Profiling metagenomic communities using circular consensus and Single Molecule, Real-Time Sequencing

There are many sequencing-based approaches to understanding complex metagenomic communities, spanning targeted amplification to whole-sample shotgun sequencing. While targeted approaches provide valuable data at low sequencing depth, they are limited by primer design and PCR amplification. Whole-sample shotgun experiments require a high depth of coverage. As such, rare community members may not be represented in the resulting assembly. Circular-consensus, Single Molecule, Real-Time (SMRT) Sequencing reads in the 1-2 kb range, with >99% consensus accuracy, can be efficiently generated for low amounts of input DNA, e.g. as little as 10 ng of input DNA sequenced in 4 SMRT Cells can generate…

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Tuesday, June 1, 2021

SMRT Sequencing of the alala genome

Single Molecule Real-Time (SMRT) Sequencing was used to generate long reads for whole genome shotgun sequencing of the genome of the`alala (Hawaiian crow). The ‘alala is endemic to Hawaii, and the only surviving lineage of the crow family, Corvidae, in the Hawaiian Islands. The population declined to less than 20 individuals in the 1990s, and today this charismatic species is extinct in the wild. Currently existing in only two captive breeding facilities, reintroduction of the ‘alala is scheduled to begin in the Fall of 2016. Reintroduction efforts will be assisted by information from the ‘alala genome generated and assembled by…

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Tuesday, June 1, 2021

Cogent: Reconstructing the coding genome from full-length transcriptome sequences

For highly complex and large genomes, a well-annotated genome may be computationally challenging and costly, yet the study of alternative splicing events and gene annotations usually rely on the existence of a genome. Long-read sequencing technology provides new opportunities to sequence full-length cDNAs, avoiding computational challenges that short read transcript assembly brings. The use of single molecule, real-time sequencing from Pacific Biosciences to sequence transcriptomes (the Iso-SeqTM method), which produces de novo, high-quality, full-length transcripts, has revealed an astonishing amount of alternative splicing in eukaryotic species. With the Iso-Seq method, it is now possible to reconstruct the transcribed regions of…

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Tuesday, June 1, 2021

Full-length cDNA sequencing for genome annotation and analysis of alternative splicing

In higher eukaryotic organisms, the majority of multi-exon genes are alternatively spliced. Different mRNA isoforms from the same gene can produce proteins that have distinct properties and functions. Thus, the importance of understanding the full complement of transcript isoforms with potential phenotypic impact cannot be understated. While microarrays and other NGS-based methods have become useful for studying transcriptomes, these technologies yield short, fragmented transcripts that remain a challenge for accurate, complete reconstruction of splice variants. The Iso-Seq protocol developed at PacBio offers the only solution for direct sequencing of full-length, single-molecule cDNA sequences to survey transcriptome isoform diversity useful for…

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Tuesday, June 1, 2021

Long-read assembly of the Aedes aegypti Aag2 cell line genome resolves ancient endogenous viral elements

Transmission of arboviruses such as Dengue Virus by Aedes aegypti causes debilitating disease across the globe. Disease in humans can include severe acute symptoms such as hemorrhagic fever and organ failure, but mosquitoes tolerate high titers of virus in a persistent infection. The mechanisms responsible for this viral tolerance are unclear. Recent publications highlighted the integration of genetic material from non-retroviral RNA viruses into the genome of the host during infection that relies upon endogenous retro-transcriptase activity from transposons. These endogenous viral elements (EVEs) found in the genome are predicted to be ancient, and at least some EVEs are under…

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Tuesday, June 1, 2021

Targeted sequencing of genes from soybean using NimbleGen SeqCap EZ and PacBio SMRT Sequencing

Full-length gene capture solutions offer opportunities to screen and characterize structural variations and genetic diversity to understand key traits in plants and animals. Through a combined Roche NimbleGen probe capture and SMRT Sequencing strategy, we demonstrate the capability to resolve complex gene structures often observed in plant defense and developmental genes spanning multiple kilobases. The custom panel includes members of the WRKY plant-defense-signaling family, members of the NB-LRR disease-resistance family, and developmental genes important for flowering. The presence of repetitive structures and low-complexity regions makes short-read sequencing of these genes difficult, yet this approach allows researchers to obtain complete sequences…

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Tuesday, June 1, 2021

An update on goat genomics

Goats are specialized in dairy, meat and fiber production, being adapted to a wide range of environmental conditions and having a large economic impact in developing countries. In the last years, there have been dramatic advances in the knowledge of the structure and diversity of the goat genome/transcriptome and in the development of genomic tools, rapidly narrowing the gap between goat and related species such as cattle and sheep. Major advances are: 1) publication of a de novo goat genome reference sequence; 2) Development of whole genome high density RH maps, and; 3) Design of a commercial 50K SNP array.…

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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

A comprehensive study of the sugar pine (Pinus lambertiana) transcriptome implemented through diverse next-generation sequencing approaches

The assembly, annotation, and characterization of the sugar pine (Pinus lambertiana Dougl.) transcriptome represents an opportunity to study the genetic mechanisms underlying resistance to the invasive white pine blister rust (Cronartium ribicola) as well as responses to other abiotic stresses. The assembled transcripts also provide a resource to improve the genome assembly. We selected a diverse set of tissues allowing the first comprehensive evaluation of the sugar pine gene space. We have combined short read sequencing technologies (Illumina MiSeq and HiSeq) with the relatively new Pacific Biosciences Iso-Seq approach. From the 2.5 billion and 1.6 million Illumina and PacBio (46…

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Tuesday, June 1, 2021

From Sequencing to Chromosomes: New de novo assembly and scaffolding methods improve the goat reference genome

Single-molecule sequencing is now routinely used to assemble complete, high-quality microbial genomes, but these assembly methods have not scaled well to large genomes. To address this problem, we previously introduced the MinHash Alignment Process (MHAP) for overlapping single-molecule reads using probabilistic, locality-sensitive hashing. Integrating MHAP with Celera Assembler (CA) has enabled reference-grade assemblies of model organisms, revealing novel heterochromatic sequences and filling low-complexity gap sequences in the GRCh38 human reference genome. We have applied our methods to assemble the San Clemente goat genome. Combining single-molecule sequencing from Pacific Biosciences and BioNano Genomics generates and assembly that is over 150-fold more…

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