scholarly journals SARS-CoV-2 genome sequencing with Oxford Nanopore Technology and Rapid PCR Barcoding in Bolivia

2021 ◽  
Author(s):  
Oscar M. Rollano-Penaloza ◽  
Carmen Delgado ◽  
Aneth Vasquez
Keyword(s):  
Developing Countries ◽  
Genome Sequencing ◽  
Amplicon Sequencing ◽  
Turnaround Time ◽  
Library Preparation ◽  
Rapid Pcr ◽  
Dna Ligation ◽  
Oxford Nanopore ◽  
Golden Standard ◽  
Average Sequencing Depth

SARS-CoV-2 genomic surveillance has Illumina technology as the golden standard. However, Oxford Nanopore Technology (ONT) provides significant improvements in accessibility, turnaround time and portability. Characteristics that gives developing countries the opportunity to perform genome surveillance. The most used protocol to sequence SARS-CoV-2 with ONT is an amplicon-sequencing protocol provided by the ARTIC Network which requires DNA ligation. Ligation reagents can be difficult to obtain in countries like Bolivia. Thus, here we provide an alternative for library preparation using the rapid PCR barcoding kit (ONT). We mapped more than 3.9 million sequence reads that allowed us to sequence twelve SARS-CoV-2 genomes from three different Bolivian cities. The average sequencing depth was 324X and the average genome length was 29527 bp. Thus, we could cover in average a 98,7% of the reference genome. The twelve genomes were successfully assigned to four different nextstrain clades (20A, 20B, 20E and 20G) and we could observe two main lineages of SARS-CoV-2 circulating in Bolivia. Therefore, this alternative library preparation for SARS-CoV-2 genome sequencing is effective to identify SARS-CoV-2 variants with high accuracy and without the need of DNA ligation. Hence, providing another tool to perform SARS-CoV-2 genome surveillance in developing countries.

scholarly journals A common protocol for the simultaneous processing of multiple clinically relevant bacterial species for whole genome sequencing

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Kathy E. Raven ◽  
Sophia T. Girgis ◽  
Asha Akram ◽  
Beth Blane ◽  
Danielle Leek ◽  
...  
Keyword(s):  
Whole Genome Sequencing ◽  
Dna Extraction ◽  
Genome Sequencing ◽  
Clinical Microbiology ◽  
Bacterial Species ◽  
Outbreak Detection ◽  
Whole Genome ◽  
Library Preparation ◽  
Simultaneous Processing ◽  
Antimicrobial Resistance Gene

AbstractWhole-genome sequencing is likely to become increasingly used by local clinical microbiology laboratories, where sequencing volume is low compared with national reference laboratories. Here, we describe a universal protocol for simultaneous DNA extraction and sequencing of numerous different bacterial species, allowing mixed species sequence runs to meet variable laboratory demand. We assembled test panels representing 20 clinically relevant bacterial species. The DNA extraction process used the QIAamp mini DNA kit, to which different combinations of reagents were added. Thereafter, a common protocol was used for library preparation and sequencing. The addition of lysostaphin, lysozyme or buffer ATL (a tissue lysis buffer) alone did not produce sufficient DNA for library preparation across the species tested. By contrast, lysozyme plus lysostaphin produced sufficient DNA across all 20 species. DNA from 15 of 20 species could be extracted from a 24-h culture plate, while the remainder required 48–72 h. The process demonstrated 100% reproducibility. Sequencing of the resulting DNA was used to recapitulate previous findings for species, outbreak detection, antimicrobial resistance gene detection and capsular type. This single protocol for simultaneous processing and sequencing of multiple bacterial species supports low volume and rapid turnaround time by local clinical microbiology laboratories.

scholarly journals Impact of Rapid PCR Influenza Testing on the Rate of Inpatient Admissions During Influenza Season at a Tertiary-Care Center

2020 ◽  
Vol 41 (S1) ◽  
pp. s263-s264
Author(s):  
Jordan Polistico ◽  
Avnish Sandhu ◽  
Teena Chopra ◽  
Erin Goldman ◽  
Jennifer LeRose ◽  
...  
Keyword(s):  
Influenza Season ◽  
Care Center ◽  
Tertiary Care ◽  
The United States ◽  
Turnaround Time ◽  
Care Hospital ◽  
Tertiary Care Center ◽  
Pediatric Hospital ◽  
Rapid Pcr ◽  
The Impact

Background: Influenza causes a high burden of disease in the United States, with an estimate of 960,000 hospitalizations in the 2017–2018 flu season. Traditional flu diagnostic polymerase chain reaction (PCR) tests have a longer (24 hours or more) turnaround time that may lead to an increase in unnecessary inpatient admissions during peak influenza season. A new point-of-care rapid PCR assays, Xpert Flu, is an FDA-approved PCR test that has a significant decrease in turnaround time (2 hours). The present study sought to understand the impact of implementing a new Xpert Flu test on the rate of inpatient admissions. Methods: A retrospective study was conducted to compare rates of inpatient admissions in patients tested with traditional flu PCR during the 2017–2018 flu season and the rapid flu PCR during the 2018–2019 flu season in a tertiary-care center in greater Detroit area. The center has 1 pediatric hospital (hospital A) and 3 adult hospitals (hospital B, C, D). Patients with influenza-like illness who presented to all 4 hospitals during 2 consecutive influenza seasons were analyzed. Results: In total, 20,923 patients were tested with either the rapid flu PCR or the traditional flu PCR. Among these, 14,124 patients (67.2%) were discharged from the emergency department and 6,844 (32.7%) were admitted. There was a significant decrease in inpatient admissions in the traditional flu PCR group compared to the rapid flu PCR group across all hospitals (49.56% vs 26.6% respectively; P < .001). As expected, a significant proportion of influenza testing was performed in the pediatric hospital, 10,513 (50.2%). A greater reduction (30% decrease in the rapid flu PCR group compared to the traditional flu PCR group) was observed in inpatient admissions in the pediatric hospital (Table 1) Conclusions: Rapid molecular influenza testing can significantly decrease inpatient admissions in a busy tertiary-care hospital, which can indirectly lead to improved patient quality with easy bed availability and less time spent in a private room with droplet precautions. Last but not the least, this testing method can certainly lead to lower healthcare costs.Funding: NoneDisclosures: None

Generating 1200bp tiled Amplicons for SARS-CoV2 Whole Genome Sequencing v2

2022 ◽  
Author(s):  
jason.nguyen not provided ◽  
Tracy Lee ◽  
Rebecca Hickman ◽  
Natalie Prystajecky ◽  
John Tyson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Oxford Nanopore ◽  
Sequencing Platforms

This procedure provides instructions for how to generate amplicons across the entire SARS-CoV-2 genome to be used for downstream whole genome sequencing applications, including Illumina MiSeq/NextSeq or Oxford Nanopore MinION sequencing platforms. The steps involved in this protocol were derived from version 3 of Freed et al protocol nCoV-2019 sequencing protocol (RAPID barcoding, 1200bp amplicon)V.3 available at https://dx.doi.org/10.17504/protocols.io.bgggjttw

scholarly journals Assessment of the scientific-technological production in molecular biology in Brazil (1996-2007): the contribution of genomics programs

2011 ◽  
Vol 83 (2) ◽  
pp. 745-760
Author(s):  
Rogério Meneghini ◽  
Estêvão C. Gamba
Keyword(s):  
Developing Countries ◽  
Molecular Biology ◽  
Genome Sequencing ◽  
Web Of Science ◽  
Scientific Production ◽  
The State ◽  
Technological Innovations ◽  
Genomic Technology ◽  
Technological Production ◽  
State Of Art

Several genome sequencing programs were launched in Brazil by the end of the nineties and the early 2000s.The most important initiatives were supported by the ONSA program (http://watson.fapesp.br/onsa/Genoma3.htm) and aimed at gaining domain in genomic technology and bringing molecular biology to the state of art. Two mainsets of data were collected in the 1996-2007 period to evaluate the results of these genome programs: the scientific production (Scopus and Web of Science databases) and the register of patents (US Patent and Trademark Office), both related to the progress of molecular biology along this period. In regard to the former, Brazil took a great leap in comparison to 17 other developed and developing countries, being only surpassed by China. As to the register of patents in the area of molecular biology, Brazil's performance lags far behind most of the countries focused in the presentstudy, confirming the Brazilian long-standing tendency of poor achievements in technological innovations when compared with scientific production. Possible solutions to surpass this inequality are discussed.

scholarly journals Evaluation of the microbial community structure of potable water samples from occupied and unoccupied buildings using16S rRNA amplicon sequencing

2020 ◽  
Author(s):  
Kimothy L Smith ◽  
Howard A Shuman ◽  
Douglas Findeisen
Keyword(s):  
Microbial Community ◽  
16S Rrna ◽  
Water Samples ◽  
Ad Hoc ◽  
Microbial Community Composition ◽  
Amplicon Sequencing ◽  
The Other ◽  
Water Usage ◽  
16S Rrna Amplicon Sequencing ◽  
Oxford Nanopore

AbstractWe conducted two studies of water samples from buildings with normal occupancy and water usage compared to water from buildings that were unoccupied with little or no water usage due to the COVID-19 shutdown. Study 1 had 52 water samples obtained ad hoc from buildings in four metropolitan locations in different states in the US and a range of building types. Study 2 had 36 water samples obtained from two buildings in one metropolitan location with matched water sample types. One of the buildings had been continuously occupied, and the other substantially vacant for approximately 3 months. All water samples were analyzed using 16S rRNA amplicon sequencing with a MinION from Oxford Nanopore Technologies. More than 127 genera of bacteria were identified, including genera with members that are known to include more than 50 putative frank and opportunistic pathogens. While specific results varied among sample locations, 16S rRNA amplicon abundance and the diversity of bacteria were higher in water samples from unoccupied buildings than normally occupied buildings as was the abundance of sequenced amplicons of genera known to include pathogenic bacterial members. In both studies Legionella amplicon abundance was relatively small compared to the abundance of the other bacteria in the samples. Indeed, when present, the relative abundance of Legionella amplicons was lower in samples from unoccupied buildings. Legionella did not predominate in any of the water samples and were found, on average, in 9.6% of samples in Study 1 and 8.3% of samples in Study 2.SynopsisComparison of microbial community composition in the plumbing of occupied and unoccupied buildings during the COVID-19 pandemic shutdown.

scholarly journals Nanopore amplicon sequencing reveals molecular convergence and local adaptation of opsin genes

2020 ◽  
Author(s):  
Katherine M. Eaton ◽  
Moisés A. Bernal ◽  
Nathan J.C. Backenstose ◽  
Trevor J. Krabbenhoft
Keyword(s):  
Local Adaptation ◽  
Environmental Gradients ◽  
Parallel Evolution ◽  
Amplicon Sequencing ◽  
Species Flock ◽  
Closely Related Species ◽  
Oxford Nanopore ◽  
Opsin Genes ◽  
Potential Case ◽  
Species Specific

AbstractLocal adaptation can drive diversification of closely related species across environmental gradients and promote convergence of distantly related taxa that experience similar conditions. We examined a potential case of adaptation to novel visual environments in a species flock (Great Lakes salmonids, genus Coregonus) using a new amplicon genotyping protocol on the Oxford Nanopore Flongle. Five visual opsin genes were amplified for individuals of C. artedi, C. hoyi, C. kiyi, and C. zenithicus. Comparisons revealed species-specific differences in the coding sequence of rhodopsin (Tyr261Phe substitution), suggesting local adaptation by C. kiyi to the blue-shifted depths of Lake Superior. Parallel evolution and “toggling” at this amino acid residue has occurred several times across the fish tree of life, resulting in identical changes to the visual systems of distantly related taxa across replicated environmental gradients. Our results suggest that ecological differences and local adaptation to distinct visual environments are strong drivers of both evolutionary parallelism and diversification.

Rapid, high throughput library preparation of SARS-CoV2 using Illumina’s DNA Prep Library Preparation Kit v2

2022 ◽  
Author(s):  
Jason Nguyen ◽  
Rebecca Hickman ◽  
Tracy Lee ◽  
Natalie Prystajecky ◽  
John Tyson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
High Throughput ◽  
Illumina Miseq ◽  
Whole Genome ◽  
Library Preparation ◽  
Dna Libraries ◽  
Reaction Volumes ◽  
Single Size ◽  
Stop Buffer

This procedure provides instructions on how to prepare DNA libraries for whole genome sequencing on an Illumina MiSeq or NextSeq using Illumina’s DNA Prep Library Preparation Kit scaled to half reaction volumes with modifications to the post-PCR procedures; tagmentation stop buffer and associated washes are removed and libraries are pooled post PCR then a single size selection is performed. This protocol is used to sequence SARS-CoV-2 using the cDNA/PCR protocol: https://dx.doi.org/10.17504/protocols.io.b3viqn4e

scholarly journals Draft Genome Sequences of Six Strains Isolated from the InSight Spacecraft and Associated Surfaces Using Oxford Nanopore- and Illumina-Based Sequencing

2020 ◽  
Vol 9 (21) ◽  
Author(s):  
Daniel L. Vera ◽  
Arman Seuylemezian ◽  
Kyle S. Landry ◽  
Ryan Hendrickson
Keyword(s):  
Whole Genome Sequencing ◽  
Genome Sequencing ◽  
Draft Genome ◽  
Whole Genome ◽  
Genome Sequences ◽  
Planetary Protection ◽  
Functional Capabilities ◽  
Oxford Nanopore ◽  
Associated Surfaces ◽  
Insight Mission

ABSTRACT Whole-genome sequencing and annotation have allowed planetary protection engineers to assess the functional capabilities of microorganisms isolated from spacecraft hardware and associated surfaces. Here, we report draft genomes of six strains isolated from the InSight mission, determined using Oxford Nanopore- and Illumina-based sequencing.

scholarly journals Constructing a Reference Genome in a Single Lab: The Possibility to Use Oxford Nanopore Technology

Plants ◽  
2019 ◽  
Vol 8 (8) ◽  
pp. 270 ◽  
Author(s):  
Yun Gyeong Lee ◽  
Sang Chul Choi ◽  
Yuna Kang ◽  
Kyeong Min Kim ◽  
Chon-Sik Kang ◽  
...  
Keyword(s):  
Plant Species ◽  
Genome Sequencing ◽  
Reference Genome ◽  
Genome Structure ◽  
Plant Genome ◽  
Sequence Information ◽  
Sequencing Analysis ◽  
Oxford Nanopore ◽  
A Genome ◽  
Long Read

The whole genome sequencing (WGS) has become a crucial tool in understanding genome structure and genetic variation. The MinION sequencing of Oxford Nanopore Technologies (ONT) is an excellent approach for performing WGS and it has advantages in comparison with other Next-Generation Sequencing (NGS): It is relatively inexpensive, portable, has simple library preparation, can be monitored in real-time, and has no theoretical limits on reading length. Sorghum bicolor (L.) Moench is diploid (2n = 2x = 20) with a genome size of about 730 Mb, and its genome sequence information is released in the Phytozome database. Therefore, sorghum can be used as a good reference. However, plant species have complex and large genomes when compared to animals or microorganisms. As a result, complete genome sequencing is difficult for plant species. MinION sequencing that produces long-reads can be an excellent tool for overcoming the weak assembly of short-reads generated from NGS by minimizing the generation of gaps or covering the repetitive sequence that appears on the plant genome. Here, we conducted the genome sequencing for S. bicolor cv. BTx623 while using the MinION platform and obtained 895,678 reads and 17.9 gigabytes (Gb) (ca. 25× coverage of reference) from long-read sequence data. A total of 6124 contigs (covering 45.9%) were generated from Canu, and a total of 2661 contigs (covering 50%) were generated from Minimap and Miniasm with a Racon through a de novo assembly using two different tools and mapped assembled contigs against the sorghum reference genome. Our results provide an optimal series of long-read sequencing analysis for plant species while using the MinION platform and a clue to determine the total sequencing scale for optimal coverage that is based on various genome sizes.

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