Comparative study on three viral enrichment approaches based on RNA extraction for plant virus/viroid detection using high-throughput sequencing

Abstract Background High-throughput sequencing (HTS) has been applied successfully for virus and viroid discovery in many agricultural crops leading to the current drive to apply this technology in routine pathogen detection. The validation of HTS-based pathogen detection is therefore paramount. Methods Plant infections were established by graft inoculating a suite of viruses and viroids from established sources for further study. Four plants (one healthy plant and three infected) were sampled in triplicate and total RNA was extracted using two different methods (CTAB extraction protocol and the Zymo Research Quick-RNA Plant Miniprep Kit) and sent for Illumina HTS. One replicate sample of each plant for each RNA extraction method was also sent for HTS on an Ion Torrent platform. The data were evaluated for biological and technical variation focussing on RNA extraction method, platform used and bioinformatic analysis. Results The study evaluated the influence of different HTS protocols on the sensitivity, specificity and repeatability of HTS as a detection tool. Both extraction methods and sequencing platforms resulted in significant differences between the data sets. Using a de novo assembly approach, complemented with read mapping, the Illumina data allowed a greater proportion of the expected pathogen scaffolds to be inferred, and an accurate virome profile was constructed. The complete virome profile was also constructed using the Ion Torrent data but analyses showed that more sequencing depth is required to be comparative to the Illumina protocol and produce consistent results. The CTAB extraction protocol lowered the proportion of viroid sequences recovered with HTS, and the Zymo Research kit resulted in more variation in the read counts obtained per pathogen sequence. The expression profiles of reference genes were also investigated to assess the suitability of these genes as internal controls to allow for the comparison between samples across different protocols. Conclusions This study highlights the need to measure the level of variation that can arise from the different variables of an HTS protocol, from sample preparation to data analysis. HTS is more comprehensive than any assay previously used, but with the necessary validations and standard operating procedures, the implementation of HTS as part of routine pathogen screening practices is possible.

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High Throughput Sequencing For Plant Virus Detection and Discovery

Phytopathology ◽

10.1094/phyto-07-18-0257-rvw ◽

2019 ◽

Vol 109 (5) ◽

pp. 716-725 ◽

Cited By ~ 44

Author(s):

D. E. V. Villamor ◽

T. Ho ◽

M. Al Rwahnih ◽

R. R. Martin ◽

I. E. Tzanetakis

Keyword(s):

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing ◽

Virus Detection ◽

Agricultural Crops ◽

Virus Discovery ◽

Plant Virus Detection ◽

Virus Biology ◽

Disease Causality

Over the last decade, virologists have discovered an unprecedented number of viruses using high throughput sequencing (HTS), which led to the advancement of our knowledge on the diversity of viruses in nature, particularly unraveling the virome of many agricultural crops. However, these new virus discoveries have often widened the gaps in our understanding of virus biology; the forefront of which is the actual role of a new virus in disease, if any. Yet, when used critically in etiological studies, HTS is a powerful tool to establish disease causality between the virus and its host. Conversely, with globalization, movement of plant material is increasingly more common and often a point of dispute between countries. HTS could potentially resolve these issues given its capacity to detect and discover. Although many pipelines are available for plant virus discovery, all share a common backbone. A description of the process of plant virus detection and discovery from HTS data are presented, providing a summary of the different pipelines available for scientists’ utility in their research.

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Current impact and future directions of high throughput sequencing in plant virus diagnostics

Virus Research ◽

10.1016/j.virusres.2014.03.029 ◽

2014 ◽

Vol 188 ◽

pp. 90-96 ◽

Cited By ~ 108

Author(s):

Sebastien Massart ◽

Antonio Olmos ◽

Haissam Jijakli ◽

Thierry Candresse

Keyword(s):

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing ◽

Future Directions ◽

Virus Diagnostics ◽

Current Impact

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Characteristics of soil nematode community under different vegetation restoration approaches in the mountainous region of southern Ningxia: A comparative study based on morphological identification and high-throughput sequencing methods

Biodiversity Science ◽

10.17520/biods.2021124 ◽

2021 ◽

Vol 29 (11) ◽

pp. 1513-1529

Author(s):

Nan Wang ◽

Jinghua Huang ◽

Na Huo ◽

Panpan Yang ◽

Xinyue Zhang ◽

...

Keyword(s):

Comparative Study ◽

High Throughput ◽

High Throughput Sequencing ◽

Vegetation Restoration ◽

Nematode Community ◽

Morphological Identification ◽

Soil Nematode ◽

Mountainous Region ◽

Soil Nematode Community ◽

Southern Ningxia

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Evaluation of direct metagenomics and target enriched approaches for high-throughput sequencing of field rabies viruses

Journal of Veterinary Research ◽

10.2478/jvetres-2019-0067 ◽

2019 ◽

Vol 63 (4) ◽

pp. 471-479

Author(s):

Anna Orłowska ◽

Ewelina Iwan ◽

Marcin Smreczak ◽

Jerzy Rola

Keyword(s):

High Throughput ◽

Deep Sequencing ◽

High Throughput Sequencing ◽

De Novo ◽

High Reliability ◽

Rna Extraction ◽

Genetic Material ◽

Target Enrichment ◽

Red Foxes ◽

Field Samples

AbstractIntroductionHigh-throughput sequencing (HTS) identifies random viral fragments in environmental samples metagenomically. High reliability gains it broad application in virus evolution, host-virus interaction, and pathogenicity studies. Deep sequencing of field samples with content of host genetic material and bacteria often produces insufficient data for metagenomics and must be preceded by target enrichment. The main goal of the study was the evaluation of HTS for complete genome sequencing of field-case rabies viruses (RABVs).Material and MethodsThe material was 23 RABVs isolated mainly from red foxes and one European bat lyssavirus-1 isolate propagated in neuroblastoma cells. Three methods of RNA isolation were tested for the direct metagenomics and RABV-enriched approaches. Deep sequencing was performed with a MiSeq sequencer (Illumina) and reagent v3 kit. Bioinformatics data were evaluated by Kraken and Centrifuge software and de novo assembly was done with metaSPAdes.ResultsTesting RNA extraction procedures revealed the deep sequencing scope superiority of the combined TRIzol/column method. This HTS methodology made it possible to obtain complete genomes of all the RABV isolates collected in the field. Significantly greater rates of RABV genome coverages (over 5,900) were obtained with RABV enrichment. Direct metagenomic studies sequenced the full length of 6 out of 16 RABV isolates with a medium coverage between 1 and 71.ConclusionDirect metagenomics gives the most realistic illustration of the field sample microbiome, but with low coverage. For deep characterisation of viruses, e.g. for spatial and temporal phylogeography during outbreaks, target enrichment is recommended as it covers sequences much more completely.

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All in One High Quality Genomic DNA and Total RNA Extraction From Nematode Induced Galls for High Throughput Sequencing Purposes

Frontiers in Plant Science ◽

10.3389/fpls.2019.00657 ◽

2019 ◽

Vol 10 ◽

Author(s):

Ana Cláudia Silva ◽

Virginia Ruiz-Ferrer ◽

Ángela Martínez-Gómez ◽

Marta Barcala ◽

Carmen Fenoll ◽

...

Keyword(s):

High Throughput ◽

Genomic Dna ◽

High Throughput Sequencing ◽

Rna Extraction ◽

High Quality ◽

Total Rna ◽

Total Rna Extraction

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A comparison of DNA/RNA extraction protocols for high-throughput sequencing of microbial communities

10.1101/2020.11.13.370387 ◽

2020 ◽

Author(s):

Justin P. Shaffer ◽

Clarisse Marotz ◽

Pedro Belda-Ferre ◽

Cameron Martino ◽

Stephen Wandro ◽

...

Keyword(s):

Microbial Community ◽

High Throughput ◽

High Throughput Sequencing ◽

Limit Of Detection ◽

Sequence Data ◽

Rna Virus ◽

Microbial Community Composition ◽

Rna Extraction ◽

Acid Extraction ◽

Dna And Rna

AbstractOne goal among microbial ecology researchers is to capture the maximum amount of information from all organisms in a sample. The recent COVID-19 pandemic, caused by the RNA virus SARS-CoV-2, has highlighted a gap in traditional DNA-based protocols, including the high-throughput methods we previously established as field standards. To enable simultaneous SARS-CoV-2 and microbial community profiling, we compare the relative performance of two total nucleic acid extraction protocols and our previously benchmarked protocol. We included a diverse panel of environmental and host-associated sample types, including body sites commonly swabbed for COVID-19 testing. Here we present results comparing the cost, processing time, DNA and RNA yield, microbial community composition, limit of detection, and well-to-well contamination, between these protocols.Accession numbersRaw sequence data were deposited at the European Nucleotide Archive (accession#: ERP124610) and raw and processed data are available at Qiita (Study ID: 12201). All processing and analysis code is available on GitHub (github.com/justinshaffer/Extraction_test_MagMAX).Methods summaryTo allow for downstream applications involving RNA-based organisms such as SARS-CoV-2, we compared the two extraction protocols designed to extract DNA and RNA against our previously established protocol for extracting only DNA for microbial community analyses. Across 10 diverse sample types, one of the two protocols was equivalent or better than our established DNA-based protocol. Our conclusion is based on per-sample comparisons of DNA and RNA yield, the number of quality sequences generated, microbial community alpha- and beta-diversity and taxonomic composition, the limit of detection, and extent of well-to-well contamination.

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High-Throughput Sequencing Facilitates Characterization of a “Forgotten” Plant Virus: The Case of a Henbane Mosaic Virus Infecting Tomato

Frontiers in Microbiology ◽

10.3389/fmicb.2018.02739 ◽

2018 ◽

Vol 9 ◽

Cited By ~ 1

Author(s):

Anja Pecman ◽

Denis Kutnjak ◽

Nataša Mehle ◽

Magda Tušek Žnidarič ◽

Ion Gutiérrez-Aguirre ◽

...

Keyword(s):

Mosaic Virus ◽

High Throughput ◽

Plant Virus ◽

High Throughput Sequencing

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Simultaneous RNA purification and size selection using on-chip isotachophoresis with an ionic spacer

Lab on a Chip ◽

10.1039/c9lc00311h ◽

2019 ◽

Vol 19 (16) ◽

pp. 2741-2749 ◽

Cited By ~ 1

Author(s):

Crystal M. Han ◽

David Catoe ◽

Sarah A. Munro ◽

Ruba Khnouf ◽

Michael P. Snyder ◽

...

Keyword(s):

High Throughput ◽

High Throughput Sequencing ◽

Rna Extraction ◽

Size Selection ◽

Rna Purification ◽

On Chip

We present an on-chip method that achieves simultaneous RNA extraction and size selection, and demonstrate its compatibility with high-throughput sequencing.

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