期刊
NEW PHYTOLOGIST
卷 228, 期 3, 页码 1149-1158出版社
WILEY
DOI: 10.1111/nph.16731
关键词
high-throughput sequencing; Illumina MiSeq; ITS; metabarcoding; metabarcoding biases; mock community; PacBio
资金
- Swedish University of Agricultural Sciences (SLU)
- Department of Forest Mycology and Plant Pathology, SLU
- Swedish Research Council's FORMAS [2011-1747]
- VR [2015-04411]
- RFI/VR (Swedish Research Council)
- Science for Life Laboratory, Sweden
- Swedish Research Council [2015-04411] Funding Source: Swedish Research Council
Recent studies have questioned the use of high-throughput sequencing of the nuclear ribosomal internal transcribed spacer (ITS) region to derive a semi-quantitative representation of fungal community composition. However, comprehensive studies that quantify biases occurring during PCR and sequencing of ITS amplicons are still lacking. We used artificially assembled communities consisting of 10 ITS-like fragments of varying lengths and guanine-cytosine (GC) contents to evaluate and quantify biases during PCR and sequencing with Illumina MiSeq, PacBio RS II and PacBio Sequel I technologies. Fragment length variation was the main source of bias in observed community composition relative to the template, with longer fragments generally being under-represented for all sequencing platforms. This bias was three times higher for Illumina MiSeq than for PacBio RS II and Sequel I. 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. Fragment length bias also increased linearly with increasing number of PCR cycles but could be mitigated by optimization of the PCR setup. No significant biases related to GC content were observed. Despite lower sequencing output, PacBio sequencing was better able to reflect the community composition of the template than Illumina MiSeq sequencing.
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