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

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Article	Current Trends in Diagnostics of Viral Infections of Unknown Etiology (DOI: 10.3390/v12020211)
Sections in this Publication
Section	Section 1. Introduction (from DOI: 10.3390/v12020211)
Section	Section 2. Traditional Methods of Diagnosing Infections (from DOI: 10.3390/v12020211)
Section	Section 3. Studying Viral Pathogens with High Throughput Sequencing (HTS) (from DOI: 10.3390/v12020211)
Section	Section 3.1. Metagenomic Approach (from DOI: 10.3390/v12020211)
Section	Section 3.2. Problems of Metagenomic Approach (from DOI: 10.3390/v12020211)
Section	Section 3.3. Methods for Improving Sequencing Output (from DOI: 10.3390/v12020211)
Section	Section 3.3.1. Nucleic Acids Depletion (from DOI: 10.3390/v12020211)
Section	Section 3.3.2. Hybridization-Based Enrichment (from DOI: 10.3390/v12020211)
Section	Section 3.3.3. Target Amplification (from DOI: 10.3390/v12020211)
Section	Section 3.4. Whole Viral Genome Sequencing (from DOI: 10.3390/v12020211)
Section	Section 3.5. Methods of Sequencing Data Analysis (from DOI: 10.3390/v12020211)
Section	Section 4. Long Read Sequencing (from DOI: 10.3390/v12020211)
Section	Section 5. Obstacles to Overcome in the Nearest Future (from DOI: 10.3390/v12020211)
Section	Section 6. Conclusions (from DOI: 10.3390/v12020211)
Section	Author Contributions (from DOI: 10.3390/v12020211)
Section	Funding (from DOI: 10.3390/v12020211)
Section	Conflicts of Interest (from DOI: 10.3390/v12020211)
Section	References (from DOI: 10.3390/v12020211)
Named Entities in this Section

From publication: "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

Section 3.3.1. Nucleic Acids Depletion

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).

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.

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.

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.

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.