View source for Section 3.3.3. Target Amplification (from DOI: 10.3390/v12020211)

{{infobox_publicationsection}}
<b>From publication:</b> " Current Trends in Diagnostics of Viral Infections of Unknown Etiology" published as Viruses; 2020 Feb 14 ; 12 (2); DOI: https://doi.org/10.1038/ 10.3390/v12020211 <br><h3><u>Section 3.3.3. Target Amplification</u></h3><p>Target PCR is an effective and simple alternative to metagenomics, working well for both individual genes and small viral genomes. PCR-based amplification is often used for whole viral genome sequencing of samples with low viral load, e.g., during the investigation of the measles outbreak during the Olympics in 2010 and epidemics of Ebola and Zika diseases. Sequencing of long (2.5-3.0 kb) amplified fragments has clarified the variability of a Norovirus genome and its spreading among patients in a few hospitals in Vietnam. A similar approach has been utilized to measure the specificity and sensitivity of the Illumina platform for detection of minor polymorphisms in mixed HIV populations. Deep sequencing of PCR-amplified viral genomes yielded complete genome sequences for the influenza virus, Dengue virus and HCV.</p><p>This approach proves apt for investigating small viral genomes that can be covered with only a few PCR amplicons. However, vast heterogeneity of RNA viruses (e.g., HCV27, Noroviruses and Rhabdoviruses) might require the use of multiple primer sets to ensure amplification of all known genotypes. Some researchers propose that this method, coupled with NGS, is used for large viral genomes, like HCMV. In this case, PCR allows long sequences to be "split" into shorter overlapping fragments that can be assembled into a full sequence during data analysis, which increases sequencing depth.</p><p>According to Reyes and Kim (1991), another type of amplification is SISPA, a method that serves well when "nucleotide sequence of the desired molecule is both unknown and present in limited amounts making its recovery by standard cloning procedures technically difficult." Recently, it has been used for quick and reliable identification and characterization of viruses.</p><p>As stated above, PCR works perfectly for amplification of small viral genomes, like those of HIV and influenza virus. For that purpose, primers are designed so that they cover a whole genome, either in a few fragments or in a single molecule. In theory, cloning shorter sequences is simpler, more reliable and increases sequencing depth, whilst also requiring compilation of primer panels for each genus. Nevertheless, target amplification has its own shortcomings. For instance, it has low data scalability, while requiring a relatively large amount of input material to ensure proper site coverage without biases. For this reason, its application is mainly restricted to samples that meet robust requirements (amount of input DNA, material quality, absence of PCR inhibitors, etc.). For instance, amplification of the whole Ebola virus genome utilizes 11 or 19 primer pairs, covering over 97% of the full sequence. Two experiments studying Noroviruses included amplification with 14 and 22 primer pairs, respectively. Finally, sequencing of the Paramurshir virus genome was conducted with a set of 60 PCR reactions and additional Sanger sequencing to cover the unamplified fragments.</p><p>Apparently, sometimes amplification, as described above, overloads laboratory workflow with numerous reactions and mandatory normalization of the products' concentrations. There is always room for error, e.g., primers failing to anneal to targets due to unmatching primer sequences (particularly in rapidly mutating viruses). It is also important to consider the costs of primer synthesis and reagents, along with the amount of labor required for setting up multiple reactions per each sample. Thus, even though this method allows for amplification and further sequencing of large viral genomes, technical complexity and low cost-effectiveness render it inapt for massive clinical research, reserving it primarily for scientific purposes.</p><p>Multiple PCRs require more input material. In clinical practice, an amount of sample drawn from a patient is usually limited. Multiplex PCR allows for parallel amplification of target sequences in a single tube, thus utilizing a smaller sample volume. In this case, compatibility of primers has to be assessed beforehand using bioinformatic tools to avoid artifacts, such as primer-dimers and false priming.</p><p>Target PCR becomes a challenge when organisms with high genome mutability are studied, e.g., HCV, influenza virus or Noroviruses. Frequent changes in their genetic sequences interfere with primer annealing. A more subtle approach to designing primers could improve the outcome, but PCR alone is by definition incapable of identifying new viruses.</p><p>We suppose that new methods based on metabarcoding might aid identification of viruses in the future. Originally, barcoding:another type of target amplification-mediated approach:was developed to assess bacterial associations (e.g., gut microbiota), based on reliable universal phylogenetic markers, such as 16S, where short taxon-specific sequences consisting of a variable fragment flanked by conservative fragments are used for tagging organisms. Metabarcoding combines a metagenomic approach (i.e., studying multiple organisms in a sample) with barcoding, making it a powerful tool for studying complex microbial associations, fungi and eukaryotes. Usually, the scalability of a metabarcoding approach is limited by genus level; however, this limitation is compensated for by a significant cost reduction in comparison with metagenomics. Unfortunately, no such loci have been described for viruses, owing primarily to their genetic diversity; however, some attempts have been made at solving this issue with broad-range primer panels, created by computational methods with promising results.</p>