**1. Introduction**

After four decades of intense efforts from all relevant fields across the world, HIV/AIDS remains a significant global public health concern. According to the Joint United Nations Programme on HIV/AIDS (UNAIDS), approximately 38 million people were living with HIV and an estimated 1.7 million people were newly infected with HIV in 2020 worldwide [1,2]. UNAIDS has set ambitious targets for the elimination of HIV/AIDS by 2030 [3]. The UNAIDS 95-95-95 targets stipulate that 95% of people living with HIV (PLWH) should be aware of their HIV status, 95% of people who are aware of their status should be receiving treatment, and 95% of people on treatment should be virally suppressed [3]. Likewise, hepatitis C virus (HCV) is another major bloodborne pathogen of significant public health concern. An estimated 58 million people currently live with chronic HCV infection, and

**Citation:** Munyuza, C.; Ji, H.; Lee, E.R. Probe Capture Enrichment Methods for HIV and HCV Genome Sequencing and Drug Resistance Genotyping. *Pathogens* **2022**, *11*, 693. https://doi.org/10.3390/ pathogens11060693

Academic Editor: Wenyu Lin

Received: 27 April 2022 Accepted: 14 June 2022 Published: 16 June 2022

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approximately 1.5 million new HCV infections occur each year [4]. In 2016, the World Health Organization (WHO) developed the Global Health Sector Strategy on Viral Hepatitis. This strategy aims to treat 80% of HCV infections, reduce new viral hepatitis infections by 90%, and reduce deaths caused by viral hepatitis infection by 65% by 2030 [4]. HIV and HCV share commonalities in that both are enveloped viruses with a positive-sense, single-stranded RNA genome. In addition, both viruses are featured by their significant genetic diversity, resulting largely from their rapid replication rates and the error-prone reverse transcriptases they rely on [5–7]. Effective HIV and HCV strain and drug resistance monitoring facilitated by genome sequencing and drug resistance (DR) genotyping help monitor the progress towards these elimination targets.

Conventional Sanger sequencing has been the primary technology applied in genome sequencing and genotypic DR testing for HIV and HCV [8,9]. Since 2005, next-generation sequencing (NGS) technologies have revolutionized the sequencing methodology, with significantly improved scalability, data throughput, sensitivity for minority resistant variants, and cost-effectiveness when batched sample testing is performed [10–13]. Nevertheless, the concentration and integrity of the input viral RNA or DNA templates determine the success of viral genotyping, regardless of the sequencing technology applied. Low viral load (VL) and low integrity often pose a significant challenge when sequencing samples collected from patients on antiviral therapy or those with severe RNA degradation [14,15].

Probe capture enrichment (also called target enrichment sequencing or hybridization capture) is a fairly recent methodology used to sequence samples containing low genomic copy numbers of a particular pathogen versus the host or from samples that have been compromised [16]. Hybridization capture involves the hybridization of sequence-specific DNA or RNA probes to a target fragment of DNA [17]. Probes are often custom-designed, targeting specific regions of interest within the template genome. For example, HIV probes can be designed to capture all major subtypes or to target particular subtypes such as subtype B of HIV-1 [18,19]. This method when performed prior to NGS would allow for complete genomes to be reconstructed directly from clinical samples. Whole genome sequencing data could then have various applications such as phylogenetics, epidemiology, and drug resistance testing [16]. Implementing this method in clinical diagnostic settings would have a direct effect on patient care as the information provided can guide patient treatment plans.

This promising method has been used successfully in a wide array of pathogens, including the parasite *Plasmodium falciparum* [20], fungi such as *Candida albicans* [21], bacteria such as *Mycobacterium tuberculosis* [22], and *Chlamydia trachomatis* [23] and viral pathogens such as HIV [18,19,24–29], HCV [24,28] and SARS-CoV-2 [30]; however, currently there is no consensus /standardized target enrichment protocol for HIV or HCV [16,31]. In this review, the various aspects of probe capture enrichment protocols used on HIV, and in some cases HCV, will be presented.

#### **2. Overview of Experimental Methods**

Hybridization capture protocols all include the same general steps [32]. The first step is nucleic acid extraction from a sample (DNA and/or RNA). This is followed by library preparation which will differ depending on the target organism, the quality and quantity of sample, and the library preparation kit being used. Target enrichment will occur after the library preparation. This process involves steps to hybridize the probes to the target sequence, enrich the probe-target complex, and elution to obtain the enriched fragment of interest. PCR-amplification will then be conducted to prepare the NGS library before sequencing on an NGS platform. The NGS data can then be processed using a professional bioinformatics platform for further analysis and alignment of the reads (Figure 1).

**Figure 1.** Overview of target-enrichment NGS procedure.

## **3. Extraction Method**

Sequencing projects typically begin by extracting nucleic acid from a given sample. The steps involved in the extraction of DNA or RNA include cell lysis, removal of membrane lipids (or other nucleic acids), purification, and concentration of the nucleic acid [33]. The most common methodologies for nucleic acid extraction include full automation or manually conducted kits. Target enrichment protocols mainly use spin columns or an automated liquid-handling robot. These two nucleic acid extraction methods were evaluated for their advantages and disadvantages by N. Ali et al. [33]. They found that column-based nucleic acid extraction is one of the best techniques used as it is fast and its results are easily reproducible. The main drawback is that it requires a small centrifuge that can generate aerosols and lead to a slight chance of cross-contamination. Conversely, automated liquid handling robots offer precise handling of reagents and samples, reducing sample loss and artificial errors. However, the main drawback to this method would be the high cost of the equipment.

The nucleic acid extraction methods used in some target enrichment protocols are summarized in Table 1. Another consideration involved in nucleic acid extraction is the starting material. Nucleic acid extraction from whole blood, plasma, and serum is typically more successful than extraction from dried blood spots (DBS) [33–35]. With its easiness of sample collection and relieved requirements for transportation and storage, DBS is becoming a popular, cost-effective alternative to plasma, serum, or whole blood for HIV-1 genotyping and VL monitoring in resource-limited settings [15,36]. However, one primary limitation of DBS for such molecular assays is that the nucleic acid integrity can be significantly compromised, making downstream PCR amplification difficult [15,36]. Although further studies are warranted, the probe capture methodology could be a solution to salvage samples of poor viral RNA integrity for molecular assays.


**Table 1.** Summary of nucleic acid extraction methods used in reported target enrichment protocols.

A successful library can be prepared from nucleic acid extracted from various sample types with either a manual or an automated protocol. Therefore, the primary considerations in choosing an extraction protocol for target enrichment will depend upon cost expectations and the availability of the required equipment.

#### **4. Library Preparation Method**

For any NGS-based project, the library preparation method is of utmost importance. The ability to generate a high-quality library is necessary for obtaining successful sequencing data. NGS library preparation is when the DNA fragments are prepared for sequencing via the addition of specific adapter sequences onto the ends of the DNA fragments (Figure 2) [37]. Several different library preparation kits and protocols can be used to produce a library, some of which are compiled in Table 2. While these kits may differ regarding their particular protocol and the amount of sample input required, most kits involve enzymatical or mechanical DNA fragmentation followed by tagmentation and incorporation of adapter sequences to the ends of the fragments. The derived libraries are then amplified and quantified prior to sequencing.

**Figure 2.** Overview of the library preparation process.



The fragmentation step is vital to the target enrichment process as it influences its outcome. Shorter fragments are captured with higher specificity than longer pieces [38]. An additional consideration when selecting a library preparation kit for target enrichment is the number of PCR amplification steps. PCR amplification can introduce bias when DNA fragments are not all amplified with the same efficiency. A negative influence of PCR amplification on the uniformity of enrichment was noted in a study conducted by Mamanova et al. [38]. This negative influence was due to the bias introduced in PCRs before and after hybridization.

Fragments that are either G-C rich or A-T rich are often underrepresented in the library preparations in comparison to G-C neutral fragments, which are amplified more efficiently [37]. One possible solution to this issue could be eliminating the PCR amplification step before hybridization, thus preventing the introduction of bias. However, while this may be possible when dealing with intact DNA available in large quantities, it lacks robustness in low-integrity samples [38]. As a result, this could be a concern when dealing

with samples such as DBS, which may contain viral templates of low integrity, rendering the PCR amplification step inevitable. A mitigation solution in such cases could be to reduce the number of PCR cycles rather than remove the step entirely in order to reduce some of the bias while also generating a robust library from low integrity samples [38]. Additionally, Van Dijk et al. [37] have suggested several library preparation methods for reducing bias in NGS, including the use of Kapa HiFi polymerase instead of the standard Phusion polymerase used in Illumina library preparation.
