scholarly journals Direct sequencing of small genomes on the Pacific Biosciences RS without library preparation

BioTechniques ◽  
2012 ◽  
Vol 53 (6) ◽  
Author(s):  
Paul Coupland ◽  
Tamir Chandra ◽  
Mike Quail ◽  
Wolf Reik ◽  
Harold Swerdlow
Keyword(s):  
Direct Sequencing ◽  
Library Preparation ◽  
Pacific Biosciences ◽  
The Pacific

The role of the mussel Mytilus spp. in the transmission of ostreid herpesvirus-1 microVar

Parasitology ◽  
2017 ◽  
Vol 145 (8) ◽  
pp. 1095-1104 ◽  
Author(s):  
A. J. O’ Reilly ◽  
C. Laide ◽  
A Maloy ◽  
S. Hutton ◽  
B. Bookelaar ◽  
...  
Keyword(s):  
Direct Sequencing ◽  
Animal Health ◽  
Viral Transmission ◽  
Diagnostic Methods ◽  
The Pacific ◽  
Herpesvirus 1 ◽  
World Organization ◽  
Ostreid Herpesvirus 1 ◽  
The Impact

AbstractThe Pacific oyster Crassostrea gigas contributes significantly to global aquaculture; however, C. gigas culture has been affected by ostreid herpesvirus-1 (OsHV-1) and variants. The dynamics of how the virus maintains itself at culture sites is unclear and the role of carriers, reservoirs or hosts is unknown. Both wild and cultured mussels Mytilus spp. (Mytilus edulis, Mytilus galloprovincialis and hybrids) are commonly found at C. gigas culture sites. The objective of this study was to investigate if Mytilus spp. can harbour the virus and if viral transmission can occur between mussels and oysters. Mytilus spp. living at oyster trestles, 400–500 m higher up the shore from the trestles and up to 26 km at non-culture sites were screened for OsHV-1 and variants by all the World Organization for Animal Health (OIE) recommended diagnostic methods including polymerase chain reaction (PCR), quantitative PCR (qPCR), histology, in situ hybridization and confirmation using direct sequencing. The particular primers that target OsHV-1 and variants, including OsHV-1 microVar (μVar), were used in the PCR and qPCR. OsHV-1 μVar was detected in wild Mytilus spp. at C. gigas culture sites and more significantly the virus was detected in mussels at non-culture sites. Cohabitation of exposed wild mussels and naïve C. gigas resulted in viral transmission after 14 days, under an elevated temperature regime. These results indicate that mussels can harbour OsHV-1 μVar; however, the impact of OsHV-1 μVar on Mytilus spp. requires further investigation.

scholarly journals Erratum: Corrigendum: Long-read sequencing of the human cytomegalovirus transcriptome with the pacific biosciences RSII platform

2018 ◽  
Vol 5 (1) ◽  
Author(s):  
Zsolt Balázs ◽  
Dóra Tombácz ◽  
Attila Szűcs ◽  
Michael Snyder ◽  
Zsolt Boldogkői
Keyword(s):  
Human Cytomegalovirus ◽  
Pacific Biosciences ◽  
The Pacific ◽  
Long Read

scholarly journals High-Quality Whole-Genome Sequence of an Estradiol-Degrading Strain, Novosphingobium tardaugens NBRC 16725

2019 ◽  
Vol 8 (11) ◽  
Author(s):  
J. Ibero ◽  
D. Sanz ◽  
B. Galán ◽  
E. Díaz ◽  
J. L. García
Keyword(s):  
Genome Sequence ◽  
Complete Sequence ◽  
Whole Genome Sequence ◽  
Whole Genome ◽  
High Quality ◽  
Content Type ◽  
Pacific Biosciences ◽  
Degrading Bacterium ◽  
The Pacific

In this work we report the complete sequence and assembly of the estradiol-degrading bacterium Novosphingobium tardaugens NBRC 16725 genome into a single contig using the Pacific Biosciences RS II system.

scholarly journals Barcoding and demultiplexing Oxford Nanopore native RNA sequencing reads with deep residual learning

2019 ◽  
Author(s):  
Martin A. Smith ◽  
Tansel Ersavas ◽  
James M. Ferguson ◽  
Huanle Liu ◽  
Morghan C Lucas ◽  
...  
Keyword(s):  
Neural Network ◽  
Rna Sequencing ◽  
Direct Sequencing ◽  
Dna Barcode ◽  
Cost Effective ◽  
Library Preparation ◽  
Rna Molecules ◽  
Base Calling ◽  
Dna Oligonucleotides ◽  
Novel Strategy

ABSTRACTNanopore sequencing has enabled sequencing of native RNA molecules without conversion to cDNA, thus opening the gates to a new era for the unbiased study of RNA biology. However, a formal barcoding protocol for direct sequencing of native RNA molecules is currently lacking, limiting the efficient processing of multiple samples in the same flowcell. A major limitation for the development of barcoding protocols for direct RNA sequencing is the error rate introduced during the base-calling process, especially towards the 5’ and 3’ ends of reads, which complicates sequence-based barcode demultiplexing. Here, we propose a novel strategy to barcode and demultiplex direct RNA sequencing nanopore data, which does not rely on base-calling or additional library preparation steps. Specifically, custom DNA oligonucleotides are ligated to RNA transcripts during library preparation. Then, raw current signal corresponding to the DNA barcode is extracted and transformed into an array of pixels, which is used to determine the underlying barcode using a deep convolutional neural network classifier. Our method,DeePlexiCon, implements a 20-layer residual neural network model that can demultiplex 93% of the reads with 95.1% specificity, or 60% of reads with 99.9% specificity. The availability of an efficient and simple barcoding strategy for native RNA sequencing will enhance the use of direct RNA sequencing by making it more cost-effective to the entire community. Moreover, it will facilitate the applicability of direct RNA sequencing to samples where the RNA amounts are limited, such as patient-derived samples.

scholarly journals Direct oligonucleotide sequencing with nanopores

2021 ◽  
Vol 1 ◽  
pp. 47
Author(s):  
Sachin Chalapati ◽  
Conor A Crosbie ◽  
Dixita Limbachiya ◽  
Nimesh Pinnamaneni
Keyword(s):  
Data Storage ◽  
Direct Sequencing ◽  
Quality Analysis ◽  
Library Preparation ◽  
Base Level ◽  
Oxford Nanopore ◽  
Sequencing Method ◽  
Annealing Step ◽  
Novel Applications ◽  
Synthesis Yield

Third-generation DNA sequencing has enabled sequencing of long, unamplified DNA fragments with minimal steps. Direct sequencing of ssDNA or RNA gives valuable insights like base-level modifications, phosphoramidite synthesis yield estimates and strand quality analysis, without the need to add the complimentary strand. Direct sequencing of single-stranded nucleic acid species is challenging as they are non-compatible to the double-stranded sequencing adapters used by manufacturers. The MinION platform from Oxford Nanopore Technologies performs sequencing by passing single-strands of DNA through a layer of biological nanopore sensors; although sequencing is performed on single-strands, the recommended template by the manufacturer is double-stranded. We have identified that the MinION platform can perform sequencing of short, single-strand oligonucleotides directly without amplification or second-strand synthesis by performing a single annealing step before library preparation. Short 5’ phosphorylated oligos when annealed to an adapter sequence can be directly sequenced in the 5' to 3' direction via nanopores. Adapter sequences were designed to bind to the 5’ end of the oligos and to leave a 3’ adenosine overhang after binding to their target. The 3’ adenosine overhang of the adapter and the terminal phosphate makes the 5’ end of the oligo analogous to an end-prepared dsDNA, rendering it compatible with ligation-based library preparation for sequencing. An oligo-pool containing 42,000, 120 nt orthogonal sequences was phosphorylated and sequenced using this method and ~90% of these sequences were recovered with high accuracy using BLAST. In the nanopore raw data, we have identified that empty signals can be wrongly identified as a valid read by the MinION platform and sometimes multiple signals containing several strands can be fused into a single raw sequence file due to segmentation faults in the software. This direct oligonucleotide sequencing method enables novel applications in DNA data storage systems where short oligonucleotides are the primary information carriers.

scholarly journals Exploring DNA structures in real-time polymerase kinetics using Pacific Biosciences sequencer data

2013 ◽  
Author(s):  
Sterling Sawaya ◽  
James Boocock ◽  
Mik Black ◽  
Neil Gemmell
Keyword(s):  
Real Time ◽  
Dna Sequences ◽  
Double Helix ◽  
Dna Structure ◽  
R Package ◽  
Dna Structures ◽  
Pacific Biosciences ◽  
Tandem Repeat Sequences ◽  
The Pacific ◽  
Polymerase Pausing

Pausing of DNA polymerase can indicate the presence of a DNA structure that differs from the canonical double-helix. Here we detail a method to investigate how polymerase pausing in the Pacific Biosciences sequencer reads can be related to DNA structure. The Pacific Biosciences sequencer uses optics to view a polymerase and its interaction with a single DNA molecule in real-time, offering a unique way to detect potential alternative DNA structures. We have developed a new way to examine polymerase kinetics and relate it to the DNA sequence by using a wavelet transform of read information from the sequencer. We use this method to examine how polymerase kinetics are related to nucleotide base composition. We then examine tandem repeat sequences known for their ability to form different DNA structures: (CGG)n and (CG)n repeats which can, respectively, form G-quadruplex DNA and Z-DNA. We find pausing around the (CGG)n repeat that may indicate the presence of G-quadruplexes in some of the sequencer reads. The (CG)n repeat does not appear to cause polymerase pausing, but its kinetics signature nevertheless suggests the possibility that alternative nucleotide conformations may sometimes be present. We discuss the implications of using our method to discover DNA sequences capable of forming alternative structures. The analyses presented here can be reproduced on any Pacific Biosciences kinetics data for any DNA pattern of interest using an R package that we have made publicly available.

scholarly journals Preparation of next-generation DNA sequencing libraries from ultra-low amounts of input DNA: Application to single-molecule, real-time (SMRT) sequencing on the Pacific Biosciences RS II.

2014 ◽  
Author(s):  
Castle Raley ◽  
David Munroe ◽  
Kristie Jones ◽  
Yu-Chih Tsai ◽  
Yan Guo ◽  
...  
Keyword(s):  
Dna Sequencing ◽  
Single Molecule ◽  
Smrt Sequencing ◽  
High Input ◽  
Single Molecule Sequencing ◽  
Pacific Biosciences ◽  
Preparation Conditions ◽  
Pacbio Rs Ii ◽  
The Pacific ◽  
The Common

We have developed and validated an amplification-free method for generating DNA sequencing libraries from very low amounts of input DNA (500 picograms - 20 nanograms) for single-molecule sequencing on the Pacific Biosciences (PacBio) RS II sequencer. The common challenge of high input requirements for single-molecule sequencing is overcome by using a carrier DNA in conjunction with optimized sequencing preparation conditions and re-use of the MagBead-bound complex. Here we describe how this method can be used to produce sequencing yields comparable to those generated from standard input amounts, but by using 1000-fold less starting material.

scholarly journals A Novel Neorickettsial Infection in 3 Dogs in the Pacific Northwest

2020 ◽  
Vol 57 (2) ◽  
pp. 286-289
Author(s):  
Gabrielle Pastenkos ◽  
Kevin Snekvik ◽  
Dan Bradway ◽  
Ilaria Cerchiaro ◽  
Susan Mehain ◽  
...  
Keyword(s):  
Pacific Northwest ◽  
Direct Sequencing ◽  
Severe Disease ◽  
Clinical Signs ◽  
Poor Response ◽  
Molecular Techniques ◽  
Response To Therapy ◽  
Cytologic Examination ◽  
The Pacific ◽  
Obligate Intracellular Bacteria

The genus Neorickettsia includes obligate, intracellular bacteria responsible for diseases including Potomac horse fever caused by Neorickettsia risticii and salmon poisoning disease (SPD) caused by Neorickettsia helminthoeca. The Stellanchasmus falcatus (SF) agent is a member of this genus previously associated only with mild clinical signs in dogs. Between 2013 and 2016, 3 dogs in Washington State (USA) presented with disease suggestive of SPD, but N. helminthoeca was not detected by molecular techniques. Clinical signs included depression, anorexia, and diarrhea. Cytologic examination of aspirates supported a diagnosis of granulomatous lymphadenitis with organisms suggestive of Neorickettsia. Dogs either died or were humanely euthanized due to poor response to therapy. Necropsy findings included lymphadenomegaly and hepatomegaly. Histopathology identified granulomatous and lymphoplasmacytic splenitis, lymphadenitis, enteritis, and hepatitis with extensive necrosis. Neorickettsia DNA was detected using genus-specific primers and direct sequencing showed 100% sequence identity to the SF agent in all 3 dogs. This is the first clinicopathologic description of severe disease in dogs attributed to the SF agent. These findings may suggest the emergence of a novel neorickettsial disease in the Pacific Northwest.

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