View source for Section 3.3.1. Nucleic Acids Depletion (from DOI: 10.3390/v12020211)

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<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/10.3390/v12020211 <br><h3><u>Section 3.3.1. Nucleic Acids Depletion</u></h3><p>Various types of clinical specimens routinely contain a large portion of host-cell nucleic acids, which can interfere with target PCR and decrease sequencing efficiency and, therefore, have to be removed in a process called depletion. Utilizing biological, physical or chemical principles, it significantly decreases the content of host-cell DNA and/or RNA, while only negligibly lowering concentration of a pathogen's nucleic acids. Most depletion kits are based on hybridization of molecular probes with target sequences, which is a lengthy but effective process. Commercial depletion kits are widely available (Ribo-Zero, RiboCop, HostZERO, NEBNext, MolYsis).</p><p>In a protocol by Gu et al., Cas9 utilizes a set of guide RNAs to target unwanted nucleic acids during depletion of abundant sequences by hybridization (DASH). Cleavage-mediated depletion resulted in an up to 99% reduction of mitochondrial DNA. This approach can be used for targeting human genomic DNA in samples with viral nucleic acids.</p><p>Despite its beneficial role in sample processing, depletion poses a few risks for the output quality. Since it acts as a bottleneck, which allows only certain sequences through, biases are inherently imposed on the selection process. Cross-hybridization is one example: recombinations in a viral genome or flaws in guide RNA design cause unspecific annealing to homologous non-target sequences, resulting in an erroneous cleavage.</p><p>Although separation of target sequences from "junk" nucleic acids is the primary goal of depletion, high levels of host-cell nucleic acids can, in theory, impede probe-sequence binding by competitive mechanisms, in which case the effectiveness of depletion would sag. However, some modified protocols have been developed that tackle this issue.</p><p>All points considered, nucleic acid depletion is an effective tool for decreasing concentrations of unwanted sequences in a sample, despite it possessing a few limitations that could lower the sequencing output by removing target nucleic acids.</p>