Section 3.3. Methods for Improving Sequencing Output (from DOI: 10.3390/v12020211)

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



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.