**4.3 Potential disadvantages and bias with 454-pyrosequencing**

Amplicon-based pyrosequencing methods have major advantages over the tools that have been used in the past to study microbial community structure. Although the results presented in this chapter have a similar pattern as the results presented Rivera et al. (2018), it important to acknowledge certain biases that have been described for amplicon-based pyrosequencing. Even though 454 pyrosequencing has a higher resolving power than Sanger sequencing or EL-FAME analysis in 454 pyrosequencing, there are some sequencing errors and chimeras that can be retained in the datasets that can inflate the estimated richness of the sample. Bias can also occur with primer selection as the primers used can select for the most predominant DNA present in the sample underestimating the rare DNA in the sample [53]. Using inappropriate primers consequently can lead to questionable biological conclusions. Another concern is that the techniques used for processing amplicon pyrosequencing data can result in the detection of several hundred "false" OTUs, mostly at low abundance, rising the concern that species abundance can be overestimated [54]. More stringent techniques such as shotgun sequencing, Ion Torrent sequencing, and Illumina platforms have been developed that help mitigate some of the concerns with pyrosequencing, but these stringent technologies have biases of their own.
