Section 3.3. Methods for Improving Sequencing Output (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. Methods for Improving Sequencing Output

Numerous methods are widely used: affinity enrichment, filtration, ultracentrifugation and depletion of the host's nucleic acids, including protocols utilizing saponin and propidium monoazide. Depletion is a powerful tool, but potential loss of target sequences restricts its use for samples with a low content of viral nucleic acids. Their fraction is usually lost during ethanol cleaning and stems from either weak probe affinity, competitive binding, low ethanol concentration or contamination with strong bases. Additionally, listed techniques are expensive and laborious. Unspecific amplification methods, e.g., multiple displacement amplification (MDA), utilize random primers and Phi29 polymerase to increase reaction output, however not necessarily with high enrichment quality, and may lead to additional contamination, uneven fragment distribution and amplification errors, lowering the overall library quality. Sometimes depletion protocols do not effectively remove host-cell DNA. Let us look further into specific techniques for improving NGS library concentration. (Figure 2)

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Figure 2: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7077230/bin/viruses-12-00211-g002.jpg
Figure 2 caption:

Unlike metagenomics (a), target HTS takes on an extra step of hybridization enrichment (b) and/or target amplification (c), during which the concentration of selected sequences is increased.

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