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MetaGenomics Sequencing In Situ
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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 (31 August 2020) | Viewed by 89552

Special Issue Editors

Dr. Aaron S. Burton

E-Mail Website
Guest Editor
NASA Johnson Space Center, Astromaterials Research and Exploration Science, Houston, TX, USA
Interests: astrobiology; DNA and RNA sequencing; astromaterials; field ecology;
Dr. Joseph Russell

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Guest Editor
MRIGlobal, Gaithersburg, MD, 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 submissions that pass pre-check are 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 2600 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 (9 papers)

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Research

Jump to: Review

18 pages, 1902 KiB  
Article
Real-Time Culture-Independent Microbial Profiling Onboard the International Space Station Using Nanopore Sequencing
by Sarah Stahl-Rommel, Miten Jain, Hang N. Nguyen, Richard R. Arnold, Serena M. Aunon-Chancellor, Gretta Marie Sharp, Christian L. Castro, Kristen K. John, Sissel Juul, Daniel J. Turner, David Stoddart, Benedict Paten, Mark Akeson, Aaron S. Burton and Sarah L. Castro-Wallace
Genes 2021, 12(1), 106; https://0-doi-org.brum.beds.ac.uk/10.3390/genes12010106 - 16 Jan 2021
Cited by 37 | Viewed by 9423
Abstract
For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms [...] Read more.
For the past two decades, microbial monitoring of the International Space Station (ISS) has relied on culture-dependent methods that require return to Earth for analysis. This has a number of limitations, with the most significant being bias towards the detection of culturable organisms and the inherent delay between sample collection and ground-based analysis. In recent years, portable and easy-to-use molecular-based tools, such as Oxford Nanopore Technologies’ MinION™ sequencer and miniPCR bio’s miniPCR™ thermal cycler, have been validated onboard the ISS. Here, we report on the development, validation, and implementation of a swab-to-sequencer method that provides a culture-independent solution to real-time microbial profiling onboard the ISS. Method development focused on analysis of swabs collected in a low-biomass environment with limited facility resources and stringent controls on allowed processes and reagents. ISS-optimized procedures included enzymatic DNA extraction from a swab tip, bead-based purifications, altered buffers, and the use of miniPCR and the MinION. Validation was conducted through extensive ground-based assessments comparing current standard culture-dependent and newly developed culture-independent methods. Similar microbial distributions were observed between the two methods; however, as expected, the culture-independent data revealed microbial profiles with greater diversity. Protocol optimization and verification was established during NASA Extreme Environment Mission Operations (NEEMO) analog missions 21 and 22, respectively. Unique microbial profiles obtained from analog testing validated the swab-to-sequencer method in an extreme environment. Finally, four independent swab-to-sequencer experiments were conducted onboard the ISS by two crewmembers. Microorganisms identified from ISS swabs were consistent with historical culture-based data, and primarily consisted of commonly observed human-associated microbes. This simplified method has been streamlined for high ease-of-use for a non-trained crew to complete in an extreme environment, thereby enabling environmental and human health diagnostics in real-time as future missions take us beyond low-Earth orbit. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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18 pages, 11615 KiB  
Article
Takeaways from Mobile DNA Barcoding with BentoLab and MinION
by Jia Jin Marc Chang, Yin Cheong Aden Ip, Chin Soon Lionel Ng and Danwei Huang
Genes 2020, 11(10), 1121; https://0-doi-org.brum.beds.ac.uk/10.3390/genes11101121 - 24 Sep 2020
Cited by 30 | Viewed by 8151
Abstract
Since the release of the MinION sequencer in 2014, it has been applied to great effect in the remotest and harshest of environments, and even in space. One of the most common applications of MinION is for nanopore-based DNA barcoding in situ for [...] Read more.
Since the release of the MinION sequencer in 2014, it has been applied to great effect in the remotest and harshest of environments, and even in space. One of the most common applications of MinION is for nanopore-based DNA barcoding in situ for species identification and discovery, yet the existing sample capability is limited (n ≤ 10). Here, we assembled a portable sequencing setup comprising the BentoLab and MinION and developed a workflow capable of processing 32 samples simultaneously. We demonstrated this enhanced capability out at sea, where we collected samples and barcoded them onboard a dive vessel moored off Sisters’ Islands Marine Park, Singapore. In under 9 h, we generated 105 MinION barcodes, of which 19 belonged to fresh metazoans processed immediately after collection. Our setup is thus viable and would greatly fortify existing portable DNA barcoding capabilities. We also tested the performance of the newly released R10.3 nanopore flow cell for DNA barcoding, and showed that the barcodes generated were ~99.9% accurate when compared to Illumina references. A total of 80% of the R10.3 nanopore barcodes also had zero base ambiguities, compared to 50–60% for R9.4.1, suggesting an improved homopolymer resolution and making the use of R10.3 highly recommended. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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14 pages, 5434 KiB  
Article
MinION-Based DNA Barcoding of Preserved and Non-Invasively Collected Wildlife Samples
by Adeline Seah, Marisa C.W. Lim, Denise McAloose, Stefan Prost and Tracie A. Seimon
Genes 2020, 11(4), 445; https://0-doi-org.brum.beds.ac.uk/10.3390/genes11040445 - 18 Apr 2020
Cited by 26 | Viewed by 8103
Abstract
The ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared [...] Read more.
The ability to sequence a variety of wildlife samples with portable, field-friendly equipment will have significant impacts on wildlife conservation and health applications. However, the only currently available field-friendly DNA sequencer, the MinION by Oxford Nanopore Technologies, has a high error rate compared to standard laboratory-based sequencing platforms and has not been systematically validated for DNA barcoding accuracy for preserved and non-invasively collected tissue samples. We tested whether various wildlife sample types, field-friendly methods, and our clustering-based bioinformatics pipeline, SAIGA, can be used to generate consistent and accurate consensus sequences for species identification. Here, we systematically evaluate variation in cytochrome b sequences amplified from scat, hair, feather, fresh frozen liver, and formalin-fixed paraffin-embedded (FFPE) liver. Each sample was processed by three DNA extraction protocols. For all sample types tested, the MinION consensus sequences matched the Sanger references with 99.29%–100% sequence similarity, even for samples that were difficult to amplify, such as scat and FFPE tissue extracted with Chelex resin. Sequencing errors occurred primarily in homopolymer regions, as identified in previous MinION studies. We demonstrate that it is possible to generate accurate DNA barcode sequences from preserved and non-invasively collected wildlife samples using portable MinION sequencing, creating more opportunities to apply portable sequencing technology for species identification. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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10 pages, 1208 KiB  
Article
Off Earth Identification of Bacterial Populations Using 16S rDNA Nanopore Sequencing
by Aaron S. Burton, Sarah E. Stahl, Kristen K. John, Miten Jain, Sissel Juul, Daniel J. Turner, Eoghan D. Harrington, David Stoddart, Benedict Paten, Mark Akeson and Sarah L. Castro-Wallace
Genes 2020, 11(1), 76; https://0-doi-org.brum.beds.ac.uk/10.3390/genes11010076 - 09 Jan 2020
Cited by 38 | Viewed by 10179
Abstract
The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station [...] Read more.
The MinION sequencer has made in situ sequencing feasible in remote locations. Following our initial demonstration of its high performance off planet with Earth-prepared samples, we developed and tested an end-to-end, sample-to-sequencer process that could be conducted entirely aboard the International Space Station (ISS). Initial experiments demonstrated the process with a microbial mock community standard. The DNA was successfully amplified, primers were degraded, and libraries prepared and sequenced. The median percent identities for both datasets were 84%, as assessed from alignment of the mock community. The ability to correctly identify the organisms in the mock community standard was comparable for the sequencing data obtained in flight and on the ground. To validate the process on microbes collected from and cultured aboard the ISS, bacterial cells were selected from a NASA Environmental Health Systems Surface Sample Kit contact slide. The locations of bacterial colonies chosen for identification were labeled, and a small number of cells were directly added as input into the sequencing workflow. Prepared DNA was sequenced, and the data were downlinked to Earth. Return of the contact slide to the ground allowed for standard laboratory processing for bacterial identification. The identifications obtained aboard the ISS, Staphylococcus hominis and Staphylococcus capitis, matched those determined on the ground down to the species level. This marks the first ever identification of microbes entirely off Earth, and this validated process could be used for in-flight microbial identification, diagnosis of infectious disease in a crewmember, and as a research platform for investigators around the world. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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10 pages, 4656 KiB  
Article
Entirely Off-Grid and Solar-Powered DNA Sequencing of Microbial Communities during an Ice Cap Traverse Expedition
by Glen-Oliver. F. Gowers, Oliver Vince, John-Henry Charles, Ingeborg Klarenberg, Tom Ellis and Arwyn Edwards
Genes 2019, 10(11), 902; https://0-doi-org.brum.beds.ac.uk/10.3390/genes10110902 - 07 Nov 2019
Cited by 29 | Viewed by 10838
Abstract
Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth’s surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging [...] Read more.
Microbial communities in remote locations remain under-studied. This is particularly true on glaciers and icecaps, which cover approximately 11% of the Earth’s surface. The principal reason for this is the inaccessibility of most of these areas due to their extreme isolation and challenging environmental conditions. While remote research stations have significantly lowered the barrier to studying the microbial communities on icecaps, their use has led to a bias for data collection in the near vicinity of these institutions. Here, miniaturisation of a DNA sequencing lab suitable for off-grid metagenomic studies is demonstrated. Using human power alone, this lab was transported across Europe’s largest ice cap (Vatnajökull, Iceland) by ski and sledge. After 11 days of unsupported polar-style travel, a metagenomic study of a geothermal hot spring gorge was conducted on the remote northern edge of the ice cap. This tent-based metagenomic study resulted in over 24 h of Nanopore sequencing, powered by solar power alone. This study demonstrates the ability to conduct DNA sequencing in remote locations, far from civilised resources (mechanised transport, external power supply, internet connection, etc.), whilst greatly reducing the time from sample collection to data acquisition. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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13 pages, 985 KiB  
Article
Tree Lab: Portable Genomics for Early Detection of Plant Viruses and Pests in Sub-Saharan Africa
by Laura M. Boykin, Peter Sseruwagi, Titus Alicai, Elijah Ateka, Ibrahim Umar Mohammed, Jo-Ann L. Stanton, Charles Kayuki, Deogratius Mark, Tarcisius Fute, Joel Erasto, Hilda Bachwenkizi, Brenda Muga, Naomi Mumo, Jenniffer Mwangi, Phillip Abidrabo, Geoffrey Okao-Okuja, Geresemu Omuut, Jacinta Akol, Hellen B. Apio, Francis Osingada, Monica A. Kehoe, David Eccles, Anders Savill, Stephen Lamb, Tonny Kinene, Christopher B. Rawle, Abishek Muralidhar, Kirsty Mayall, Fred Tairo and Joseph Ndunguruadd Show full author list remove Hide full author list
Genes 2019, 10(9), 632; https://0-doi-org.brum.beds.ac.uk/10.3390/genes10090632 - 21 Aug 2019
Cited by 63 | Viewed by 12590
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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14 pages, 2557 KiB  
Communication
Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS)
by Samir V. Deshpande, Timothy M. Reed, Raymond F. Sullivan, Lee J. Kerkhof, Keith M. Beigel and Mary M. Wade
Genes 2019, 10(8), 578; https://0-doi-org.brum.beds.ac.uk/10.3390/genes10080578 - 30 Jul 2019
Cited by 19 | Viewed by 7306
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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11 pages, 1745 KiB  
Article
A Rapid and Accurate MinION-Based Workflow for Tracking Species Biodiversity in the Field
by Simone Maestri, Emanuela Cosentino, Marta Paterno, Hendrik Freitag, Jhoana M. Garces, Luca Marcolungo, Massimiliano Alfano, Iva Njunjić, Menno Schilthuizen, Ferry Slik, Michele Menegon, Marzia Rossato and Massimo Delledonne
Genes 2019, 10(6), 468; https://0-doi-org.brum.beds.ac.uk/10.3390/genes10060468 - 20 Jun 2019
Cited by 100 | Viewed by 11598
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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Review

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16 pages, 1357 KiB  
Review
Genetic Biomonitoring and Biodiversity Assessment Using Portable Sequencing Technologies: Current Uses and Future Directions
by Henrik Krehenwinkel, Aaron Pomerantz and Stefan Prost
Genes 2019, 10(11), 858; https://0-doi-org.brum.beds.ac.uk/10.3390/genes10110858 - 29 Oct 2019
Cited by 61 | Viewed by 10009
Abstract
We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable [...] Read more.
We live in an era of unprecedented biodiversity loss, affecting the taxonomic composition of ecosystems worldwide. The immense task of quantifying human imprints on global ecosystems has been greatly simplified by developments in high-throughput DNA sequencing technology (HTS). Approaches like DNA metabarcoding enable the study of biological communities at unparalleled detail. However, current protocols for HTS-based biodiversity exploration have several drawbacks. They are usually based on short sequences, with limited taxonomic and phylogenetic information content. Access to expensive HTS technology is often restricted in developing countries. Ecosystems of particular conservation priority are often remote and hard to access, requiring extensive time from field collection to laboratory processing of specimens. The advent of inexpensive mobile laboratory and DNA sequencing technologies show great promise to facilitate monitoring projects in biodiversity hot-spots around the world. Recent attention has been given to portable DNA sequencing studies related to infectious organisms, such as bacteria and viruses, yet relatively few studies have focused on applying these tools to Eukaryotes, such as plants and animals. Here, we outline the current state of genetic biodiversity monitoring of higher Eukaryotes using Oxford Nanopore Technology’s MinION portable sequencing platform, as well as summarize areas of recent development. Full article
(This article belongs to the Special Issue MetaGenomics Sequencing In Situ)
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