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

ABSTRACT: High-throughput sequencing (HTS) has become increasingly popular as virus diagnostic tool. It has been used to detect and identify plant viruses and viroids in different types of matrices and tissues. A viral sequence enrichment method prior to HTS is required to increase the viral reads in the generated data to ease the bioinformatic analysis of generated sequences. In this study, we compared the sensitivity of three viral enrichment approaches, i.e. double stranded RNA (dsRNA), ribosomal RNA depleted total RNA (ribo-depleted totRNA) and small RNA (sRNA) for plant virus/viroid detection, followed by sequencing on MiSeq and NextSeq Illumina platforms. The three viral enrichment approaches used here enabled the detection of all viruses/viroid used in this study. When the data was normalised, the recovered viral/viroid nucleotides and depths were depending on the viral genome and the enrichment method used. Both dsRNA and ribo-depleted totRNA approaches detected a divergent strain of Wuhan aphid virus 2 that was not expected in this sample. Additionally, Vicia cryptic virus was detected in the data of dsRNA and sRNA approaches only. The results suggest that dsRNA enrichment has the highest potential to detect and identify plant viruses and viroids. The dsRNA approach used here detected all viruses/viroid, consumed less time, was lower in cost, and required less starting material. Therefore, this approach appears to be suitable for diagnostics laboratories.

SUBMITTER: Gaafar YZA

PROVIDER: S-EPMC7447037 | biostudies-literature | 2020

REPOSITORIES: biostudies-literature

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

Gaafar Yahya Zakaria Abdou YZA Ziebell Heiko H

PloS one 20200825 8

High-throughput sequencing (HTS) has become increasingly popular as virus diagnostic tool. It has been used to detect and identify plant viruses and viroids in different types of matrices and tissues. A viral sequence enrichment method prior to HTS is required to increase the viral reads in the generated data to ease the bioinformatic analysis of generated sequences. In this study, we compared the sensitivity of three viral enrichment approaches, i.e. double stranded RNA (dsRNA), ribosomal RNA d ...[more]

PMID: 32841302

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Project description:BackgroundThe recent development of next-generation sequencing DNA marker technologies, such as genotyping-by-sequencing (GBS), generates thousands of informative single nucleotide polymorphism markers in almost any species, regardless of genomic resources. This enables poorly resourced or "orphan" crops/species access to high-density, high-throughput marker platforms which have revolutionised population genetics studies and plant breeding. DNA quality underpins success of GBS methods as the DNA must be amenable to restriction enzyme digestion and sequencing. A barrier to implementing GBS technologies is access to inexpensive, high-throughput extraction methods that yield sequencing-quality genomic DNA (gDNA) from plants. Several high-throughput DNA extraction methods are available, but typically provide low yield or poor quality gDNA, or are costly (US$6-$9/sample) for consumables.ResultsWe modified a non-organic solvent protocol to extract microgram quantities (1-13 μg) of sequencing-quality high molecular weight gDNA inexpensively in 96-well plates from either fresh, freeze-dried or silica gel-dried plant tissue. The protocol was effective for several easy and difficult-to-extract forage, crop, horticultural and common model species including Trifolium, Medicago, Lolium, Secale, Festuca, Malus, Oryza, and Arabidopsis. The extracted DNA was of high molecular weight and digested readily with restriction enzymes. Contrasting with other extraction protocols we assessed, Illumina-based sequencing of GBS libraries developed from this gDNA had very uniform high quality base-calls to the end of sequence reads. Furthermore, DNA extracted using this method has been sequenced successfully with the PacBio long-read platform. The protocol is scalable, readily automated without requirement for fume hoods, requires approximately three hours to process 192 samples (384-576 samples/day), and is inexpensive at US$0.62/sample for consumables.ConclusionsThis versatile, scalable and simple protocol yields high molecular weight genomic DNA suitable for restriction enzyme digestion and next-generation sequencing applications including GBS and long-read sequencing platforms such as PacBio. The low cost, high-throughput, and extraction of high quality gDNA from a range of fresh and dried source plant material makes this method suitable for many sequencing and genotyping applications including large-scale sample screening underpinning breeding programmes.

Project description:Plant organellar genomes contain large, repetitive elements that may undergo pairing or recombination to form complex structures and/or sub-genomic fragments. Organellar genomes also exist in admixtures within a given cell or tissue type (heteroplasmy), and an abundance of subtypes may change throughout development or when under stress (sub-stoichiometric shifting). Next-generation sequencing (NGS) technologies are required to obtain deeper understanding of organellar genome structure and function. Traditional sequencing studies use several methods to obtain organellar DNA: (1) If a large amount of starting tissue is used, it is homogenized and subjected to differential centrifugation and/or gradient purification. (2) If a smaller amount of tissue is used (i.e., if seeds, material, or space is limited), the same process is performed as in (1), followed by whole-genome amplification to obtain sufficient DNA. (3) Bioinformatics analysis can be used to sequence the total genomic DNA and to parse out organellar reads. All these methods have inherent challenges and tradeoffs. In (1), it may be difficult to obtain such a large amount of starting tissue; in (2), whole-genome amplification could introduce a sequencing bias; and in (3), homology between nuclear and organellar genomes could interfere with assembly and analysis. In plants with large nuclear genomes, it is advantageous to enrich for organellar DNA to reduce sequencing costs and sequence complexity for bioinformatics analyses. Here, we compare a traditional differential centrifugation method with a fourth method, an adapted CpG-methyl pulldown approach, to separate the total genomic DNA into nuclear and organellar fractions. Both methods yield sufficient DNA for NGS, DNA that is highly enriched for organellar sequences, albeit at different ratios in mitochondria and chloroplasts. We present the optimization of these methods for wheat leaf tissue and discuss major advantages and disadvantages of each approach in the context of sample input, protocol ease, and downstream application.

Dataset Information

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

Publications

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

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