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