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Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19

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

Deadline for manuscript submissions: closed (15 April 2021) | Viewed by 54763

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Special Issue Editors

Dr. Miten Jain

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Guest Editor

UCSC Genomics Institute, University of California, Santa Cruz, CA 95064, USA
Interests: biotechology; bioinformatics; nanopore sequencing
Special Issues, Collections and Topics in MDPI journals

Dr. Hugh E. Olsen

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Guest Editor

UCSC Genomics Institute, University of California, Santa Cruz, CA 95064, USA
Interests: biotechnology; bioinformatics; nanopore sequencing

Prof. Dr. Ana Conesa

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Guest Editor

Microbiology and Cell Science, Institute for Food and Agricultural Research, Genetics Institute, University of Florida, Gainesville, FL 32611-0700, USA
Interests: bioinformatics; transcriptomics; multi-omics data integration; systems genomics; functional annotation; alternative splicing

Dr. Adam Ameur

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Guest Editor

Department of Immunology, Genetics and Pathology, Uppsala University, Science for Life Laboratory, 75108 Uppsala, Sweden
Interests: next-generation sequencing; single-molecule sequencing; clinical sequencing; bioinformatics; genomics
Special Issues, Collections and Topics in MDPI journals

Special Issue Information

Dear colleagues,

The COVID-19 pandemic has affected global health and the global economy at an unprecedented scale. This event has necessitated that we implement strong measures for the early detection of and rapid response to infectious diseases and potential pandemics in the immediate future.

This Issue will focus on technologies that have been developed for the detection and diagnosis of infectious diseases and viral agents. In particular, it will focus on the diagnosis and characterization of SARS-CoV-2 using different genomics and sequencing technologies.

Several groups, both academic and commercial, from across the world, have done groundbreaking research in regard to developing rapid, point-of-care diagnostic tests for SARS-CoV-2. Additionally, there is substantial work on tracking the genomic epidemiology of the virus using state-of-the-art sequencing platforms. There is also an ongoing focus on patient and population stratification studies as well as the search of patient SNPs associated to susceptibility.

As an update from 2021, we will also consider studies on host interactions, such as the genomic and transcriptomic aspects of vaccination and immunity.

We invite the research community from both academia and industry to submit their work on SARS-CoV-2 for this Issue. Even though the focus of the Issue is on COVID-19, we encourage colleagues to submit their work on relevant infectious diseases and genomics technologies. Per se all publications will remain open access during and after the pandemic.

These research findings will reveal further insights into the COVID-19 pandemic outbreak and its effects. This Issue will help define the actions and guidelines for surveillance, containment, and mitigation, for future events of such magnitude.

Dr. Miten Jain
Dr. Hugh E. Olsen
Prof. Ana Conesa
Dr. Adam Ameur
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

Genomics technologies
Sequencing techniques
Metagenomics
Virus sequencing
Genetic diagnostics
qPCR
Nanopore sequencing
Single-molecule real-time (SMRT) sequencing
Next-generation sequencing (NGS)
Third-generation sequencing
Virus characterization
Long-read sequencing
Comparative genomics
Functional genomics
Diagnostic tests
Infectious diseases
Pandemic
SARS-CoV-2
COVID-19
Coronavirus
Genomic epidemiology
Population stratification
Genetic susceptibility
Host interactions

Published Papers (10 papers)

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Research

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12 pages, 2916 KiB

Open AccessArticle

Relative Consolidation of the Kappa Variant Pre-Dates the Massive Second Wave of COVID-19 in India

by Jitendra Singh, Anvita Gupta Malhotra, Debasis Biswas, Prem Shankar, Leena Lokhande, Ashvini Kumar Yadav, Arun Raghuvanshi, Dipesh Kale, Shashwati Nema, Saurabh Saigal and Sarman Singh

Genes 2021, 12(11), 1803; https://doi.org/10.3390/genes12111803 - 16 Nov 2021

Cited by 6 | Viewed by 2016

Abstract

India experienced a tragic second wave after the end of March 2021, which was far more massive than the first wave and was driven by the emergence of the novel delta variant (B.1.617.2) of the SARS-CoV-2 virus. In this study, we explored the local and national landscape of the viral variants in the period immediately preceding the second wave to gain insight into the mechanism of emergence of the delta variant and thus improve our understanding of the causation of the second wave. We randomly selected 20 SARS-CoV-2 positive samples diagnosed in our lab between 3 February and 8 March 2021 and subjected them to whole genome sequencing. Nine of the 20 sequenced genomes were classified as kappa variant (B.1.617.1). The phylogenetic analysis of pan-India SARS-CoV-2 genome sequences also suggested the gradual replacement of the α variant with the kappa variant during this period. This relative consolidation of the kappa variant was significant, since it shared 3 of the 4 signature mutations (L452R, E484Q and P681R) observed in the spike protein of delta variant and thus was likely to be the precursor in its evolution. This study demonstrates the predominance of the kappa variant in the period immediately prior to the second wave and underscores its role as the “bridging variant” between the α and delta variants that drove the first and second waves of COVID-19 in India, respectively. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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14 pages, 1415 KiB

Open AccessArticle

Evaluation of RT-qPCR and Loop-Mediated Isothermal Amplification (LAMP) Assays for the Detection of SARS-CoV-2 in Argentina

by María Dolores Fellner, Romina Bonaventura, Jorge Basiletti, Martín Avaro, Estefanía Benedetti, Ana Campos, María Elena Dattero, Mara Russo, Sara Vladmirsky, Viviana Molina, Lucía Irazu, Marcelo A. Rodriguez, Andrea Pontoriero, Daniel M. Cisterna and Elsa G. Baumeister

Genes 2021, 12(5), 659; https://doi.org/10.3390/genes12050659 - 28 Apr 2021

Cited by 6 | Viewed by 2658

Abstract

Our aim was to evaluate the analytical and clinical performance of the SARS-CoV-2 molecular detection kits used in Argentina. Nine real-time reverse-transcription polymerase chain reaction (RT-qPCR) and three reverse-transcription loop-mediated isothermal amplification (RT-LAMP) assays were evaluated using the World Health Organization (WHO) recommended test as reference method. A secondary standard calibrated for the E, N and RdRp genes against the Pan American Health Organization—World Health Organization—International Standard was used to calculate the limit of detection (LoD). A panel of artificial clinical samples, 32 positive and 30 negative for SARS-CoV-2, were analyzed to estimate the kappa concordance (κ) and the diagnostic performance. Differences among the LoD values for the target genes amplified by each kit were >1 log copies/reaction. The κ for the RT-qPCR kits was greater than 0.9, whereas that for the RT-LAMP assays ranged from 0.75 to 0.93. The clinical performance of RT-qPCR kits showed 100% specificity and high sensitivity, although with variations according to the gene analyzed. The E and N genes provided greater clinical sensitivity, whereas the RdRp gene increased the clinical specificity. The RT-LAMP assays revealed a variable diagnostic performance. The information provided can be useful to choose the most appropriate diagnostic test and may contribute to the establishment of a consensus in the diagnosis of SARS-CoV-2 in Argentina and the region. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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10 pages, 2249 KiB

Open AccessArticle

Genotyping of the Major SARS-CoV-2 Clade by Short-Amplicon High-Resolution Melting (SA-HRM) Analysis

by Hector Diaz-Garcia, Ana L. Guzmán-Ortiz, Tania Angeles-Floriano, Israel Parra-Ortega, Briceida López-Martínez, Mirna Martínez-Saucedo, Guillermo Aquino-Jarquin, Rocío Sánchez-Urbina, Hector Quezada and Javier T. Granados-Riveron

Genes 2021, 12(4), 531; https://doi.org/10.3390/genes12040531 - 05 Apr 2021

Cited by 14 | Viewed by 3725

Abstract

The genome of the SARS-CoV-2 virus, the causal agent of the COVID-19 pandemic, has diverged due to multiple mutations since its emergence as a human pathogen in December 2019. Some mutations have defined several SARS-CoV-2 clades that seem to behave differently in terms of regional distribution and other biological features. Next-generation sequencing (NGS) approaches are used to classify the sequence variants in viruses from individual human patients. However, the cost and relative scarcity of NGS equipment and expertise in developing countries prevent studies aimed to associate specific clades and variants to clinical features and outcomes in such territories. As of March 2021, the GR clade and its derivatives, including the B.1.1.7 and B.1.1.28 variants, predominate worldwide. We implemented the post-PCR small-amplicon high-resolution melting analysis to genotype SARS-CoV-2 viruses isolated from the saliva of individual patients. This procedure was able to clearly distinguish two groups of samples of SARS-CoV-2-positive samples predicted, according to their melting profiles, to contain GR and non-GR viruses. This grouping of the samples was validated by means of amplification-refractory mutation system (ARMS) assay as well as Sanger sequencing. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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10 pages, 1089 KiB

Open AccessEditor’s ChoiceArticle

Overcoming Supply Shortage for SARS-CoV-2 Detection by RT-qPCR

by Gustavo Barcelos Barra, Ticiane Henriques Santa Rita, Pedro Góes Mesquita, Rafael Henriques Jácomo and Lídia Freire Abdalla Nery

Genes 2021, 12(1), 90; https://doi.org/10.3390/genes12010090 - 13 Jan 2021

Cited by 15 | Viewed by 2383

Abstract

In February 2020, our laboratory started to offer a RT-qPCR assay for the qualitative detection of severe acute respiratory syndrome coronavirus 2. A few months after the assay was released to our patients, some materials, reagents, and equipment became in short supply. Alternative protocols were necessary in order to avoid stopping testing to the population. However, the suitability of these alternatives needs to be validated before their use. Here, we investigated if saliva is a reliable alternative specimen to nasopharyngeal swabs; if 0.45% saline is a reliable alternative to guanidine hydrochloride as a collection viral transport media; the stability of SARS-COV-2 in guanidine hydrochloride and in 0.45% saline for 10 and 50 days at room temperature; and if the primers/probe concentration and thermocycling times could be reduced so as to overcome the short supply of these reagents and equipment, without a significant loss of the assay performance. We found that saliva is not an appropriated specimen for our method—nasopharyngeal swabs perform better. Saline (0.45%) and guanidine hydrochloride have a similar SARS-CoV-2 diagnostic capability as tube additives. Reliable SARS-CoV-2 RNA detection can be performed after sample storage for 10 days at room temperature (18–23 °C) in both 0.45% saline and guanidine hydrochloride. Using synthetic RNA, and decreasing the concentration of primers by five-fold and probes by 2.5-fold, changed the assay limit of detection (LOD) from 7.2 copies/reaction to 23.7 copies/reaction and the subsequent reducing of thermocycling times changed the assay LOD from 23.7 copies/reaction to 44.2 copies/reaction. However, using real clinical samples with Cq values ranging from ~12.15 to ~36.46, the results of the three tested conditions were almost identical. These alterations will not affect the vast majority of diagnostics and increase the daily testing capability in 30% and increase primers and probe stocks in 500% and 250%, respectively. Taken together, the alternative protocols described here overcome the short supply of tubes, reagents and equipment during the SARS-CoV-2 pandemic, avoiding the collapse of test offering for the population: 105,757 samples were processed, and 25,156 SARS-CoV-2 diagnostics were performed from 9 May 2020 to 30 June 2020. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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14 pages, 4063 KiB

Open AccessArticle

Transcriptional Differences for COVID-19 Disease Map Genes between Males and Females Indicate a Different Basal Immunophenotype Relevant to the Disease

by Tianyuan Liu, Leandro Balzano-Nogueira, Ana Lleo and Ana Conesa

Genes 2020, 11(12), 1447; https://doi.org/10.3390/genes11121447 - 01 Dec 2020

Cited by 13 | Viewed by 3076

Abstract

Worldwide COVID-19 epidemiology data indicate differences in disease incidence amongst sex and gender demographic groups. Specifically, male patients are at a higher death risk than female patients, and the older population is significantly more affected than young individuals. Whether this difference is a consequence of a pre-existing differential response to the virus, has not been studied in detail. We created DeCovid, an R shiny app that combines gene expression (GE) data of different human tissue from the Genotype-Tissue Expression (GTEx) project along with the COVID-19 Disease Map and COVID-19 related pathways gene collections to explore basal GE differences across healthy demographic groups. We used this app to study differential gene expression of COVID-19 associated genes in different age and sex groups. We identified that healthy women show higher expression-levels of interferon genes. Conversely, healthy men exhibit higher levels of proinflammatory cytokines. Additionally, young people present a stronger complement system and maintain a high level of matrix metalloproteases than older adults. Our data suggest the existence of different basal immunophenotypes amongst different demographic groups, which are relevant to COVID-19 progression and may contribute to explaining sex and age biases in disease severity. The DeCovid app is an effective and easy to use tool for exploring the GE levels relevant to COVID-19 across demographic groups and tissues. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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14 pages, 2668 KiB

Open AccessArticle

Analytical Sensitivity and Specificity of Two RT-qPCR Protocols for SARS-CoV-2 Detection Performed in an Automated Workflow

by Gustavo Barcelos Barra, Ticiane Henriques Santa Rita, Pedro Góes Mesquita, Rafael Henriques Jácomo and Lídia Freire Abdalla Nery

Genes 2020, 11(10), 1183; https://doi.org/10.3390/genes11101183 - 12 Oct 2020

Cited by 35 | Viewed by 6026

Abstract

WHO declared the novel coronavirus (COVID-19) outbreak a global pandemic on 11 March 2020. The establishment of standardized RT-qPCR protocols for respiratory secretions testing, as well as sharing of specimens, data, and information became critical. Here, we investigate the analytical performance of two interim RT-qPCR protocols (Charité and Centers for Disease Control (CDC)) for the qualitative detection of SARS-CoV-2 executed in a fully automated platform. Analytical specificity, PCR amplification efficiency, analytical sensitivity (limit of detection), and cross-reactivity were evaluated using contrived samples. The on-going accuracy was evaluated by retrospective analysis of our test results database (real clinical samples). N1, E, and a modified version of RdRP assays presented adequate analytical specificity, amplification efficiency, and analytical sensitivity using contrived samples. The three assays were applied to all individuals who requested the SARS-CoV-2 molecular test assay in our laboratory and it was observed that N1 gave more positive results than E, and E gave more positive results than RdRP (modified). The RdRP and E were removed from the test and its final version, based on N1 assay only, was applied to 30,699 Brazilian individuals (from 19 February 2020 to 8 May 2020). The aggregated test results available in the database were also presented. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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13 pages, 1725 KiB

Open AccessEditor’s ChoiceArticle

Whole Genome Sequencing of SARS-CoV-2: Adapting Illumina Protocols for Quick and Accurate Outbreak Investigation during a Pandemic

by Sureshnee Pillay, Jennifer Giandhari, Houriiyah Tegally, Eduan Wilkinson, Benjamin Chimukangara, Richard Lessells, Yunus Moosa, Stacey Mattison, Inbal Gazy, Maryam Fish, Lavanya Singh, Khulekani Sedwell Khanyile, James Emmanuel San, Vagner Fonseca, Marta Giovanetti, Luiz Carlos Alcantara, Jr. and Tulio de Oliveira

Genes 2020, 11(8), 949; https://doi.org/10.3390/genes11080949 - 17 Aug 2020

Cited by 51 | Viewed by 12686

Abstract

The COVID-19 pandemic has spread very fast around the world. A few days after the first detected case in South Africa, an infection started in a large hospital outbreak in Durban, KwaZulu-Natal (KZN). Phylogenetic analysis of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) genomes can be used to trace the path of transmission within a hospital. It can also identify the source of the outbreak and provide lessons to improve infection prevention and control strategies. This manuscript outlines the obstacles encountered in order to genotype SARS-CoV-2 in near-real time during an urgent outbreak investigation. This included problems with the length of the original genotyping protocol, unavailability of reagents, and sample degradation and storage. Despite this, three different library preparation methods for Illumina sequencing were set up, and the hands-on library preparation time was decreased from twelve to three hours, which enabled the outbreak investigation to be completed in just a few weeks. Furthermore, the new protocols increased the success rate of sequencing whole viral genomes. A simple bioinformatics workflow for the assembly of high-quality genomes in near-real time was also fine-tuned. In order to allow other laboratories to learn from our experience, all of the library preparation and bioinformatics protocols are publicly available at protocols.io and distributed to other laboratories of the Network for Genomics Surveillance in South Africa (NGS-SA) consortium. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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14 pages, 1124 KiB

Open AccessArticle

Evaluation of the Ion AmpliSeq SARS-CoV-2 Research Panel by Massive Parallel Sequencing

by Federica Alessandrini, Sara Caucci, Valerio Onofri, Filomena Melchionda, Adriano Tagliabracci, Patrizia Bagnarelli, Laura Di Sante, Chiara Turchi and Stefano Menzo

Genes 2020, 11(8), 929; https://doi.org/10.3390/genes11080929 - 12 Aug 2020

Cited by 25 | Viewed by 4419

Abstract

Deep knowledge of the genetic features of SARS-CoV-2 is essential to track the ongoing pandemic through different geographical areas and to design and develop early diagnostic procedures, therapeutic strategies, public health interventions, and vaccines. We describe protocols and first results of the Ion AmpliSeq™ SARS-CoV-2 Research Panel by a massively parallel sequencing (MPS) assay. The panel allows for targeted sequencing by overlapping amplicons, thereby providing specific, accurate, and high throughput analysis. A modified reverse transcription reaction, which consists of the use of a SARS-CoV-2 specific primers pool from the Ion AmpliSeq SARS-CoV-2 Research Panel, was assessed in order to promote viral RNA specific reverse transcription. The aim of this study was to evaluate the effectiveness of the Ion AmpliSeq™ SARS-CoV-2 Research Panel in sequencing the entire viral genome in different samples. SARS-CoV-2 sequence data were obtained from ten viral isolates and one nasopharyngeal swab from different patients. The ten isolate samples amplified with 12 PCR cycles displayed high mean depth values compared to those of the two isolates amplified with 20 PCR cycles. High mean depth values were also obtained for the nasopharyngeal swab processed by use of a target-specific reverse transcription. The relative depth of coverage (rDoC) analysis showed that when 12 PCR cycles were used, all target regions were amplified with high sequencing coverage, while in libraries amplified at 20 cycles, a poor uniformity of amplification, with absent or low coverage of many target regions, was observed. Our results show that the Ion AmpliSeq SARS-CoV-2 Research Panel can achieve rapid and high throughput SARS-CoV-2 whole genome sequencing from 10 ng of DNA-free viral RNA from isolates and from 1 ng of DNA-free viral RNA from a nasopharyngeal swab using 12 PCR cycles for library amplification. The modified RT-PCR protocol yielded superior results on the nasopharyngeal swab compared to the reverse transcription reaction set up according to the manufacturer’s instructions. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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13 pages, 1423 KiB

Open AccessEditor’s ChoiceArticle

Rapid Direct Nucleic Acid Amplification Test without RNA Extraction for SARS-CoV-2 Using a Portable PCR Thermocycler

by Soon Keong Wee, Suppiah Paramalingam Sivalingam and Eric Peng Huat Yap

Genes 2020, 11(6), 664; https://doi.org/10.3390/genes11060664 - 18 Jun 2020

Cited by 60 | Viewed by 10989

Abstract

There is an ongoing worldwide coronavirus disease 2019 (Covid-19) pandemic caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). At present, confirmatory diagnosis is by reverse transcription polymerase chain reaction (RT-PCR), typically taking several hours and requiring a molecular laboratory to perform. There is an urgent need for rapid, simplified, and cost-effective detection methods. We have developed and analytically validated a protocol for direct rapid extraction-free PCR (DIRECT-PCR) detection of SARS-CoV-2 without the need for nucleic acid purification. As few as six RNA copies per reaction of viral nucleocapsid (N) gene from respiratory samples such as sputum and nasal exudate can be detected directly using our one-step inhibitor-resistant assay. The performance of this assay was validated on a commercially available portable PCR thermocycler. Viral lysis, reverse transcription, amplification, and detection are achieved in a single-tube homogeneous reaction within 36 min. This minimizes hands-on time, reduces turnaround-time for sample-to-result, and obviates the need for RNA purification reagents. It could enable wider use of Covid-19 testing for diagnosis, screening, and research in countries and regions where laboratory capabilities are limiting. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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9 pages, 1598 KiB

Open AccessEditor’s ChoiceBrief Report

Relative Abundance of SARS-CoV-2 Entry Genes in the Enterocytes of the Lower Gastrointestinal Tract

by Jaewon J. Lee, Scott Kopetz, Eduardo Vilar, John Paul Shen, Ken Chen and Anirban Maitra

Genes 2020, 11(6), 645; https://doi.org/10.3390/genes11060645 - 11 Jun 2020

Cited by 53 | Viewed by 5261

Abstract

There is increasing evidence of gastrointestinal (GI) infection by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). We surveyed the co-expression of SARS-CoV-2 entry genes ACE2 and TMPRSS2 throughout the GI tract to assess potential sites of infection. Publicly available and in-house single-cell RNA-sequencing datasets from the GI tract were queried. Enterocytes from the small intestine and colonocytes showed the highest proportions of cells co-expressing ACE2 and TMPRSS2. Therefore, the lower GI tract represents the most likely site of SARS-CoV-2 entry leading to GI infection. Full article

(This article belongs to the Special Issue Sequencing Techniques and Genomics Technologies to Help with Diagnostics and Virus Characterization – Focus on COVID 19)

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