Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi

Jana M. U'Ren, Jakob M. Riddle, James T. Monacell, Ignazio Carbone, Jolanta Miadlikowska, Anne E Arnold

Research output: Contribution to journalArticle

41 Citations (Scopus)

Abstract

Next-generation sequencing technologies have provided unprecedented insights into fungal diversity and ecology. However, intrinsic biases and insufficient quality control in next-generation methods can lead to difficult-to-detect errors in estimating fungal community richness, distributions and composition. The aim of this study was to examine how tissue storage prior to DNA extraction, primer design and various quality-control approaches commonly used in 454 amplicon pyrosequencing might influence ecological inferences in studies of endophytic and endolichenic fungi. We first contrast 454 data sets generated contemporaneously from subsets of the same plant and lichen tissues that were stored in CTAB buffer, dried in silica gel or freshly frozen prior to DNA extraction. We show that storage in silica gel markedly limits the recovery of sequence data and yields a small fraction of the diversity observed by the other two methods. Using lichen mycobiont sequences as internal positive controls, we next show that despite careful filtering of raw reads and utilization of current best-practice OTU clustering methods, homopolymer errors in sequences representing rare taxa artificially increased estimates of richness c. 15-fold in a model data set. Third, we show that inferences regarding endolichenic diversity can be improved using a novel primer that reduces amplification of the mycobiont. Together, our results provide a rationale for selecting tissue treatment regimes prior to DNA extraction, demonstrate the efficacy of reducing mycobiont amplification in studies of the fungal microbiomes of lichen thalli and highlight the difficulties in differentiating true information about fungal biodiversity from methodological artefacts.

Original languageEnglish (US)
Pages (from-to)1032-1048
Number of pages17
JournalMolecular Ecology Resources
Volume14
Issue number5
DOIs
StatePublished - 2014

Fingerprint

Lichens
lichen
lichens
community composition
Fungi
Silica Gel
fungus
silica gel
DNA
Quality Control
quality control
fungi
amplification
gel
silica
DNA Primers
Biodiversity
fungal communities
Ecology
thallus

Keywords

  • 454 amplicon pyrosequencing
  • Diversity
  • ITSrDNA
  • Lichen
  • Moss
  • Mycobiont
  • Peltigera
  • Pinus

ASJC Scopus subject areas

  • Ecology, Evolution, Behavior and Systematics
  • Biotechnology
  • Genetics
  • Medicine(all)

Cite this

Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi. / U'Ren, Jana M.; Riddle, Jakob M.; Monacell, James T.; Carbone, Ignazio; Miadlikowska, Jolanta; Arnold, Anne E.

In: Molecular Ecology Resources, Vol. 14, No. 5, 2014, p. 1032-1048.

Research output: Contribution to journalArticle

U'Ren, Jana M. ; Riddle, Jakob M. ; Monacell, James T. ; Carbone, Ignazio ; Miadlikowska, Jolanta ; Arnold, Anne E. / Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi. In: Molecular Ecology Resources. 2014 ; Vol. 14, No. 5. pp. 1032-1048.
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