Table 1 Microbiome methods and limitations

Bias introduced during extraction, amplification, sequencing, and bioinformatic processing can alter the relative abundances of species within a sample [44]. Relative abundances can range from 50-fold higher or lower than actual depending on the specific species contribution and protocols used [44]. The complete absence of a species may reflect bias below the limit of detection. Conversely, expansion of specific taxa may reflect progressive and systemic bias enriching for sequencing reads from those taxa [44]. It is therefore important to consider and correct for these biases in any experiment where taxon relative abundance is considered using computational methods [44]. A key step in any metagenomic sequencing experiment is to sequence similar, defined communities of different taxon proportions to understand bias in each protocol. Sequencing-defined communities can lead to computational estimates of protocol bias that can be applied to all samples prior to analysis [44]. Furthermore, extraction and processing introduce contamination depending on its format, and each kit has its own DNA that needs to be evaluated especially when considering a potentially sterile site [45,46,47]. Thus, sequencing both mock, negative controls of the sequencing kit only and contrived, defined bacterial communities is essential for optimal microbiome sequencing determination.