Distinguishing Closely Related Species: NGS may struggle to differentiate closely related species with nearly identical DNA sequences, often resulting in identification only at the genus or family level.
DNA Quality and Degradation: The presence of degraded DNA, especially in highly processed samples or those with low pH, can lead to incomplete or inconclusive species identification.
Sensitivity Issues: While NGS is highly sensitive, this can also be a drawback. Low-abundance sequences or those with unexpected lengths may be detected but could be due to PCR or sequencing errors, requiring filtering out during data analysis.
False Positives/Negatives: Due to the method's sensitivity, there is a risk of false positives if sequences present at low levels are misidentified. Conversely, species present in very low abundance (typically below 0.5% of all assigned reads) may not be reported.
Dependence on Reference Databases: The accuracy of species identification relies heavily on the completeness and quality of reference sequences in databases like NCBI. Uncharacterized or poorly annotated species may be missed.
Primer Bias: Different primer sets may amplify certain species better than others, leading to an incomplete representation of the microbial community. It’s important to choose primers carefully based on their known limitations.