Section 3.3.2. Hybridization-Based Enrichment (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 3.3.2. Hybridization-Based Enrichment
Experiments that compare modern clinical assays with HTS-based tests conclude that data derived from sequencing might be helpful in several ways. It can serve for pathogen detection and identification and also supply clinically valuable information on the presence of resistance genes or resistance-yielding mutations in a pathogen's genome. It could help healthcare professionals choose the correct therapy without having to wait for microbiological tests and prescribing broad-spectrum drugs that are constantly shown to have adverse side effects. Of note is that NGS finds pathogens beyond standard testing panels, unlike traditional tests. However, genetic libraries must be created, often from samples with low concentrations of a pathogen's nucleic acids and high content of nucleic acids from a host and other microorganisms. An elegant solution has been proposed by two independent teams of researchers, resulting in the development of a novel commercially available enrichment tool based on hybridization.
The most notable feature of this approach is that molecular probes, instead of targeting selected viral species or families, bind to certain sequences in all known viral genera infecting vertebrates, thus supposedly creating the most comprehensive tool for viral screening. This enrichment technique greatly increases sequencing depth, while also raising the fraction of viral reads by 100-10,000 times compared to standard library preparation protocols. A diagnostic tool of such sensitivity could serve as an alternative to traditional methods when they fail to detect the pathogen. In contrast, total sample sequencing could at least partly detect a new pathogen's sequences, revealing its identity.
Target probe-based enrichment (also known as hybridization-based enrichment) is used for studying viral genomes without prior cultivation or clonal amplification. For this, short DNA or RNA probes are used, which are complementary to the chosen sequences in a viral genome. The trick is to use several probes for one viral genome, thus ensuring its full coverage and guaranteed extraction: even if the genome contains major mutations, there is a distinct possibility that some target sequences remain unchanged, allowing for binding with a probe. Probes themselves are biotinylated at one end, binding strongly to beads covered in streptavidin. After that, a magnet separator is used to wash the beads from any redundant DNA and RNA with further elution of target sequences.
Hybridization-based enrichment holds a few advantages over PCR-based techniques. Alterations to a pathogen's sequence impede primer annealing, whereas probes tend to bind to target sites, given proper reaction conditions and time, even if the target sequence contains polymorphisms. Besides, hybridization works well for covering large genomes, demonstrating less coverage biases.
This approach has been successfully used to describe clinically significant viruses with varying genome sizes, such as HCV, HSV-1, VZV, EBV, HCMV, HHV6 and HHV7. Hybridization can be set up in a single tube and is easily automated. Moreover, the use of probes yields results more reliable than those after cultivation. This is because acquired sequences are almost totally identical to original templates, whereas cultivation creates quasispecies, distorting actual genetic structure of a viral population.
Sensitivity of hybridization is increased when probes are designed using a large set of reference sequences, resulting in a better coverage of all conceivable genetic variants. This is achieved because target enrichment is still possible when target sequences differ from a probe sequence. However, full sequences are needed when creating hybridization probes, while designing PCR primers requires sequences only of the flanking regions. Another advantage of probe-based methods is that if some oligonucleotides fail to anneal during hybridization, overlapping probes might compensate.
Briese et al. developed VirCapSeq-VERT:a platform for concentrating nucleic acids of vertebrate viruses. It contains approximately two million biotinylated probes for target enrichment. VirCapSeq-VERT detects virtually all human viruses, including new ones, featuring up to 40% of unknown sequences. Another team of researchers created an analogous approach, called ViroCap, which has been tested in a virological lab on actual clinical samples. In 26 samples, metagenomic shotgun sequencing (MSS) with ViroCap detected all anticipated viruses plus 30 that had not been identified before. An experiment carried out later demonstrated that out of 30 viruses identified with NGS and missed by clinical tests, 18 were identified by MSS and ViroCap, and an additional 12 with ViroCap only. Another powerful computational tool for designing probes has been recently developed by Metsky et al.. CATCH (compact aggregation of targets for comprehensive hybridization) analyzes target viral genomes and selects a minimum number of probes for optimal coverage of multiple viruses. This results in an effective hybridization-based enrichment, while preserving the original viral diversity of a sample. Reduction in the number of probes curtails experiment costs, while increasing the efficiency of metagenomic sequencing.
Nevertheless, the price per sample remains high for this approach, and it has not gained much popularity yet due to insufficient data on its relative performance. In addition, hybridization protocols are time-consuming. According to official protocols, hybridization may take up to 24 h in some cases, after which there are additional steps required for library preparation, consuming a few extra hours. Secondly, viral genomes that differ from probe sequences for more than 40% will most likely fail to undergo enrichment. Although this does not guarantee the loss of these viruses, the effectiveness of the procedure nosedives. For these reasons, MSS and PCR with degenerate primers might prove more effective, albeit yielding more "noise" data in the output, which is removable during processing. In the end, the choice of enrichment method boils down to the experiment design and quality of the input material.
