**6. Drawbacks of NSG and dPCR**

One of the most common issues affecting the efficiency of NGS and dPCR is sample quality. Although platforms are often tested and compared using highly selected samples, real-life samples do not behave in the same way. Although "next generation" massively parallel DNA sequencers have been shown to offer many potential benefits in performing genetic analyses, especially for large-scale projects, one of the main drawbacks limiting their use is the relatively low error rate highly compared to standard short-read methods. In general, the main disadvantages observed in the different existing platforms are the presence of a bias in the CG ratio, high cost, and errors in the homopolymers (substitution/deletion errors in the readings) because their length is not correctly deduced from the electrical signal, the introduction of new algorithms, post-sequencing correction tools and the SNV/insertions-deletions (INDEL) tool [128], low output, short reads, and high error rate [129]. Likewise, there are also problems with long-length reads. In general, NSG and other long-read sequencing platforms suffer from a high error rate [130]. On the other hand, accuracy stands out as the most important issue for all recently developed technologies. For example, in metagenomic whole genome sequencing (mWGS), which analyzes all of the DNA or RNA in a given sample, in addition to high cost, an additional consideration for performing mWGS is the abundance of human DNA present in many types of clinical samples, such as blood and respiratory secretions [131]. Another barrier to the implementation of NGS in the diagnosis of infectious diseases has been the time it takes to complete all the steps for NGS, this turnaround time relative to conventional methods has limited the clinical relevance of NGS results for decision-making decisions about patient care [132].

On the other hand, although dPCR has numerous advantages over qPCR, it has not yet been able to fully replace the use of qPCR. It has been reported that high concentrations of nucleic acid could saturate the dPCR reaction, highlighting the importance of adequate dilution [133–136]. The low throughput compared to qPCR and the longer response times of current dPCR systems have not allowed this technique to enter routine analysis. On the other hand, the exclusivity of reagents based on the platform used is another limitation as it does not allow working with a different platform, including the multiplexing approach. Some dPCR protocols show a dynamic range similar to or smaller than that offered by a qPCR assay, which in some cases decreases the sensitivity of the assay, and lastly, the costs of equipment and reagents are still somewhat inaccessible [74, 137].

*Metabarcoding and Digital PCR (dPCR): Application in the Study of Neglected Tropical Diseases DOI: http://dx.doi.org/10.5772/intechopen.106272*
