When degenerated primers are used, excessive PCR cycles may bias amplification towards templates matching nondepleted primers (Lindahl et al., 2013). Similarly, preferential amplification or lack of amplification of specific taxa is influenced by primer as well as marker choice (Tedersoo & Lindahl, 2016 Nilsson et al., 2019). The PCR may be biased towards preferential amplification of specific DNA templates with lower cytosine-guanine (GC) content or of shorter lengths (Benjamini & Speed, 2012 Ihrmark et al., 2012). Marker amplification by polymerase chain reaction (PCR) is recognized as one of the most important sources of community distortion in metabarcoding studies (Tedersoo et al., 2018). In addition, any remaining effects of various biases should be assessed, quantified and compensated for in downstream analyses. Therefore, it is necessary to establish laboratory protocols and data handling strategies that are optimized to preserve qualitative and quantitative representation of community members throughout DNA amplification and sequencing. Such biases and errors affect perceived differences in composition and diversity among communities, and should be minimized (Frøslev et al., 2017 Nilsson et al., 2019). correctly representing relative abundances of members) in fungal community studies has been questioned based on observations of biases and errors when sequencing artificially assembled (‘mock’) communities (Amend et al., 2010 Bakker, 2018 Palmer et al., 2018). Thus, the interpretation of internal transcribed spacer (ITS) sequencing data as semi-quantitative (i.e. However, overcoming biases and errors introduced during amplification and sequencing remains a major challenge (Nilsson et al., 2019). One of the main strengths of these technologies is that communities can be taxonomically profiled at relatively low costs and effort. The use of high-throughput DNA sequencing technologies in the field of fungal ecology has increased our understanding of how fungi affect processes in soils and ecosystems (Lindahl et al., 2013 Nilsson et al., 2019). Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.No significant biases related to GC content were observed. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. All 10 fragments in the artificial community were recovered when sequenced with PacBio technologies, whereas the three longest fragments (> 447 bases) were lost when sequenced with Illumina MiSeq. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I.
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