Importance of taking an appropriate sample

Well planned and proper sample acquisition is essential for ensuring the reliability of sequencing results. It is crucial to ensure that the sample that gets sequenced is collected and processed appropriately. The point is to ensure that the nucleic acids obtained from samples are of good quality and quantity, otherwise, it is easy to get results that are not fully representative of that sample. “A bad sample produces bad results.” Because most of the samples collected will contain mostly the host genome, it is essential to target a body site and to use a technique that captures as much of the viral genome as possible.

The following steps are worth considering when performing the sampling process.

The sample collection site

The sampling site should be chosen depending on the target organism. Therefore it is essential to collect a sample from the site where the target nucleic acids are highly abundant while ensuring that the most non-invasive procedure is the best for the patient. A case in point, if SARS-CoV-2 is the target organism, it is recommended to collect a sample from the upper respiratory tract where the organism is abundant. In a meta-analysis of the performance of samples collected from different sites, Lee et al, 2021 demonstrated different SARS-CoV-2 positivity rates from different sample types.

Sample collection procedure

The sample collection procedure impacts the quality of the sample. For respiratory viruses, nasopharyngeal swabs are the most recommended. Proper collection of the nasopharyngeal swab was reported to increase the chances of getting the virus. For example, a study by Irifune et al, 2021 about the discrepancy of SARS-CoV-2 PCR results due to the sample collection sites and possible improper sampling showed that different viral copy numbers are obtained when the sample collection technique is changed.

Sample transport media

Viral Transport Media is often recommended for sample collection to prevent bacterial growth/contamination and to keep the integrity of the sample therefore, the transport media in which the sample is collected may impact the possibility of recovering the virus. However, Banada et al, 2021 demonstrated that eNAT sterilizing buffer enhanced the sensitive detection of COVID-19 compared to the ordinary transport media. Certain media used for virus diagnostic purposes, particularly some point-of-care tests, severely impact the ability to generate high-quality sequence data.

Sample storage and preservation

The sample storage affects the quality of the sample because SARS-CoV-2 is an RNA virus, it is essential to store the samples at the most ambient temperature. Indeed some studies have demonstrated that samples with a low viral load have the potential of turning negative when stored at room temperature or 4o C within 6 days.

Sample enrichment bias

Before sequencing, some protocols amplify the SARS-CoV-2 target to decrease the amount of the host RNA. For example, the ARTIC protocol, the most commonly used method for SARS-CoV-2 sequencing has a PCR step, that may not evenly amplify all sections of the viral genome. Therefore, some parts of the viral genome may be over-amplified while others may be underrepresented. Interestingly, even the polymerase used in this PCR step should be a high fidelity one otherwise polymerases with a low fidelity may introduce mutations before sequencing.