Special Issue "MetaGenomics Sequencing In Situ"

A special issue of Genes (ISSN 2073-4425). This special issue belongs to the section "Molecular Genetics and Genomics".

Deadline for manuscript submissions: closed (1 September 2019).

Special Issue Editors

Dr. Aaron S. Burton
E-Mail Website
Guest Editor
NASA Johnson Space Center, Astromaterials Research and Exploration Science, Houston, United States
Interests: astrobiology; DNA and RNA sequencing; astromaterials; field ecology;
Dr. Joseph Russell
E-Mail
Guest Editor
MRIGlobal, Gaithersburg, Maryland, USA
Interests: nanopore sequencing; MinION; infectious disease diagnosis; field ecology; metagenomics; direct RNA sequencing; epigenetics; methylation; long reads; agriculture; biosurveillance

Special Issue Information

Dear Colleagues,

In situ sequencing has a broad range of applications, such as for infectious disease diagnosis and tracking, ecological and agricultural research, and even space exploration, including monitoring crew health and the search for extra-terrestrial life. The MinION sequencer has given the opportunity to improve in situ sequencing as it measures differences in current caused by DNA or RNA strands passing through nanopores embedded in membranes, with the change in current at a given time being diagnostic of the nucleotides passing through the pore. Because nanopore-based sequencing directly measures the molecules rather than relying on their synthesis, it can be used to sequence RNA directly without having to convert it to cDNA first, as well as detect modified bases in both DNA and RNA. The footprint and energy requirements of the MinION is also significantly smaller than other sequencing platforms, enabling use in settings outside of traditional or core laboratories.

In this Special Issue, we seek original research and reviews of field applications of nanopore sequencing, including the development of in situ sample preparation and processing hardware and procedures, as well as local data processing and analysis.

Dr. Aaron S. Burton
Dr. Joseph Russel
Guest Editors

Manuscript Submission Information

Manuscripts should be submitted online at www.mdpi.com by registering and logging in to this website. Once you are registered, click here to go to the submission form. Manuscripts can be submitted until the deadline. All papers will be peer-reviewed. Accepted papers will be published continuously in the journal (as soon as accepted) and will be listed together on the special issue website. Research articles, review articles as well as short communications are invited. For planned papers, a title and short abstract (about 100 words) can be sent to the Editorial Office for announcement on this website.

Submitted manuscripts should not have been published previously, nor be under consideration for publication elsewhere (except conference proceedings papers). All manuscripts are thoroughly refereed through a single-blind peer-review process. A guide for authors and other relevant information for submission of manuscripts is available on the Instructions for Authors page. Genes is an international peer-reviewed open access monthly journal published by MDPI.

Please visit the Instructions for Authors page before submitting a manuscript. The Article Processing Charge (APC) for publication in this open access journal is 1800 CHF (Swiss Francs). Submitted papers should be well formatted and use good English. Authors may use MDPI's English editing service prior to publication or during author revisions.

Keywords

  • nanopore sequencing
  • MinION
  • infectious disease diagnosis
  • field ecology
  • metagenomics
  • direct RNA sequencing
  • epigenetics
  • methylation
  • long reads
  • agriculture

Published Papers (3 papers)

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Research

Open AccessArticle
Tree Lab: Portable Genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa
Genes 2019, 10(9), 632; https://doi.org/10.3390/genes10090632 - 21 Aug 2019
Abstract
In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using [...] Read more.
In this case study we successfully teamed the PDQeX DNA purification technology developed by MicroGEM, New Zealand, with the MinION and MinIT mobile sequencing devices developed by Oxford Nanopore Technologies to produce an effective point-of-need field diagnostic system. The PDQeX extracts DNA using a cocktail of thermophilic proteinases and cell wall-degrading enzymes, thermo-responsive extractor cartridges and a temperature control unit. This closed system delivers purified DNA with no cross-contamination. The MinIT is a newly released data processing unit that converts MinION raw signal output into nucleotide base called data locally in real-time, removing the need for high-specification computers and large file transfers from the field. All three devices are battery powered with an exceptionally small footprint that facilitates transport and setup. To evaluate and validate capability of the system for unbiased pathogen identification by real-time sequencing in a farmer’s field setting, we analysed samples collected from cassava plants grown by subsistence farmers in three sub-Sahara African countries (Tanzania, Uganda and Kenya). A range of viral pathogens, all with similar symptoms, greatly reduce yield or destroy cassava crops. Eight hundred (800) million people worldwide depend on cassava for food and yearly income, and viral diseases are a significant constraint to its production. Early pathogen detection at a molecular level has great potential to rescue crops within a single growing season by providing results that inform decisions on disease management, use of appropriate virus-resistant or replacement planting. This case study presented conditions of working in-field with limited or no access to mains power, laboratory infrastructure, Internet connectivity and highly variable ambient temperature. An additional challenge is that, generally, plant material contains inhibitors of downstream molecular processes making effective DNA purification critical. We successfully undertook real-time on-farm genome sequencing of samples collected from cassava plants on three farms, one in each country. Cassava mosaic begomoviruses were detected by sequencing leaf, stem, tuber and insect samples. The entire process, from arrival on farm to diagnosis, including sample collection, processing and provisional sequencing results was complete in under 3 h. The need for accurate, rapid and on-site diagnosis grows as globalized human activity accelerates. This technical breakthrough has applications that are relevant to human and animal health, environmental management and conservation. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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Open AccessCommunication
Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS)
Genes 2019, 10(8), 578; https://doi.org/10.3390/genes10080578 - 30 Jul 2019
Abstract
Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this [...] Read more.
Field laboratories interested in using the MinION often need the internet to perform sample analysis. Thus, the lack of internet connectivity in resource-limited or remote locations renders downstream analysis problematic, resulting in a lack of sample identification in the field. Due to this dependency, field samples are generally transported back to the lab for analysis where internet availability for downstream analysis is available. These logistics problems and the time lost in sample characterization and identification, pose a significant problem for field scientists. To address this limitation, we have developed a stand-alone data analysis packet using open source tools developed by the Nanopore community that does not depend on internet availability. Like Oxford Nanopore Technologies’ (ONT) cloud-based What’s In My Pot (WIMP) software, we developed the offline MinION Detection Software (MINDS) based on the Centrifuge classification engine for rapid species identification. Several online bioinformatics applications have been developed surrounding ONT’s framework for analysis of long reads. We have developed and evaluated an offline real time classification application pipeline using open source tools developed by the Nanopore community that does not depend on internet availability. Our application has been tested on ATCC’s 20 strain even mix whole cell (ATCC MSA-2002) sample. Using the Rapid Sequencing Kit (SQK-RAD004), we were able to identify all 20 organisms at species level. The analysis was performed in 15 min using a Dell Precision 7720 laptop. Our offline downstream bioinformatics application provides a cost-effective option as well as quick turn-around time when analyzing samples in the field, thus enabling researchers to fully utilize ONT’s MinION portability, ease-of-use, and identification capability in remote locations. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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Open AccessArticle
A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
Genes 2019, 10(6), 468; https://doi.org/10.3390/genes10060468 - 20 Jun 2019
Abstract
Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for [...] Read more.
Genetic markers (DNA barcodes) are often used to support and confirm species identification. Barcode sequences can be generated in the field using portable systems based on the Oxford Nanopore Technologies (ONT) MinION sequencer. However, to achieve a broader application, current proof-of-principle workflows for on-site barcoding analysis must be standardized to ensure a reliable and robust performance under suboptimal field conditions without increasing costs. Here, we demonstrate the implementation of a new on-site workflow for DNA extraction, PCR-based barcoding, and the generation of consensus sequences. The portable laboratory features inexpensive instruments that can be carried as hand luggage and uses standard molecular biology protocols and reagents that tolerate adverse environmental conditions. Barcodes are sequenced using MinION technology and analyzed with ONTrack, an original de novo assembly pipeline that requires as few as 1000 reads per sample. ONTrack-derived consensus barcodes have a high accuracy, ranging from 99.8 to 100%, despite the presence of homopolymer runs. The ONTrack pipeline has a user-friendly interface and returns consensus sequences in minutes. The remarkable accuracy and low computational demand of the ONTrack pipeline, together with the inexpensive equipment and simple protocols, make the proposed workflow particularly suitable for tracking species under field conditions. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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