Comparing Viral Metagenomic Extraction Methods

Abstract

A crucial step in the molecular detection of viruses in clinical specimens is the efficient extraction of viral nucleic acids. The total yield of viral nucleic acid from a clinical specimen is dependent on the specimen's volume, the initial virus concentration and the effectiveness provided by the extraction method. Recent Next Generation Sequencing (NGS)-based diagnostic approaches (i.e. metagenomics) provide a molecular 'open view' into the sample, as they theoretically generate sequence reads of any nucleic acid present in a specimen in a statistically representative manner. However, since a higher virus-related read output promises better sensitivity in the subsequent bioinformatic analysis, the extraction method selected determines the reliability of diagnostic NGS. In this study nine commercially available kits for nucleic acid extraction were compared regarding the simultaneous isolation of DNA and RNA by real-time PCR,four of which were selected for subsequent comparison by NGS (QIAamp Viral RNA Mini Kit, QIAamp DNA Blood Mini Kit, QIAamp cador Pathogen Mini Kit and QIAamp MinElute Virus Spin Kit). The nucleic acid yields and the sequence read output were compared for four different model viruses comprising Reovirus, Orthomyxovirus, Orthopoxvirus and Paramyxovirus, each at defined but varying concentrations in the same sample. The total amount of nucleic acid was processed to sequence the RNA (as cDNA) and the DNA with quantification by Qubit and virus-specific quantitative real-time PCRs. NGS libraries were prepared for sequencing on the Illumina HiSeq 1500 system. Finally, the percentage of reads assignable to each virus was determined via mapping. Evaluation of different commercial nucleic acid extraction kits with four different viruses indicates little variation in the read numbers obtained for transcribed RNA or DNA by NGS. Since NGSis increasingly being used as a tool in diagnostics of infectious diseases, the individual steps of the complete process have to be validated carefully. Here we could show that for virus identification in liquid clinical specimens, any nucleic acid extraction kit that is performing well for PCR diagnostics can be used for NGS diagnostics as well and that the selection of the kit has only a minor impact on the yield of viral reads.

Comparison of Nucleic Acid Extraction Methods for a Viral Metagenomics Analysis of Respiratory Viruses

“…To date, few studies have assessed the impact of different extraction methods on the performance of metagenomics. Klenner et al evaluated four manual QIAamp nucleic acid extraction kits (QIAamp Viral RNA Mini Kit, QIAamp DNA Blood Mini Kit, QIAamp cador Pathogen Mini Kit, and QIAamp MinElute Virus Spin Kit) with four different viruses (including Reovirus , Orthomyxovirus , Orthopoxvirus , and Paramyxovirus ), and reported that the selection of the kits has only a minor impact on the yield of viral reads and the quantity of reads obtained by NGS [ 28 ]. However, this study only evaluated manual kits from the same manufacturer and with separate RNA and DNA extraction methods.…”

“…While there are many manual and automatic extraction methods available, it is important to choose the most sensitive and reliable one for mNGS. Numerous studies evaluating different extraction platforms in terms of their viral qPCR performance have found that the choice of extraction platform has a major impact on the reliability of the diagnostic results [ 24 , 25 , 26 , 27 , 28 , 29 ]. Furthermore, nucleic acid extraction methods can also impact bacteriome profiles [ 30 , 31 , 32 ] as well as the detection of particular viruses with mNGS [ 16 , 23 , 27 , 29 ].…”

Development of a Sensitive Real-Time Fast-qPCR Based on SYBR® Green for Detection and Quantification of Chicken Parvovirus (ChPV)

“…The assay efficiency curve was 101.94% showing that the results are highly reliable, and the standard curve can be used for an absolute quantification of the viral genome. In addition, the melting curve showed a unique peak at 79.03 °C that could be used to confirm ChPV in a sample, and therefore avoid false-negative results in tissue samples with interfering DNA, such as thymus, spleen, bursa, and blood [ 33 , 34 ]. Hence, in the present study, ChPV was detected and quantified in the enteric contents of broilers, laying hens, and breeders using the fast-qPCR based on the SYBR ® Green assay that had been reported in previous studies [ 22 ].…”

Implementation and Validation of the Roche Light Cycler 480 96-Well Plate Platform as a Real-Time PCR Assay for the Quantitative Detection of Cytomegalovirus (CMV) in Clinical Specimens Using the Luminex MultiCode ASRs System

“…Specimen DNA extraction for viral pathogen materials and human tissues was as previously described [17]. The extraction of viral RNA and the storage of sample material were critical, as viral RNA integrity affected qPCR sensitivity and specificity.…”