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

Publication ,  Journal Article
U'Ren, JM; Riddle, JM; Monacell, JT; Carbone, I; Miadlikowska, J; Arnold, AE
Published in: Molecular ecology resources
September 2014

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.

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Published In

Molecular ecology resources

DOI

EISSN

1755-0998

ISSN

1755-098X

Publication Date

September 2014

Volume

14

Issue

5

Start / End Page

1032 / 1048

Related Subject Headings

  • Specimen Handling
  • Sequence Analysis, DNA
  • Preservation, Biological
  • Plants
  • Molecular Sequence Data
  • Fungi
  • Evolutionary Biology
  • Endophytes
  • Diagnostic Errors
  • DNA Primers
 

Citation

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U’Ren, J. M., Riddle, J. M., Monacell, J. T., Carbone, I., Miadlikowska, J., & Arnold, A. E. (2014). Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi. Molecular Ecology Resources, 14(5), 1032–1048. https://doi.org/10.1111/1755-0998.12252
U’Ren, Jana M., Jakob M. Riddle, James T. Monacell, Ignazio Carbone, Jolanta Miadlikowska, and A Elizabeth Arnold. “Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi.Molecular Ecology Resources 14, no. 5 (September 2014): 1032–48. https://doi.org/10.1111/1755-0998.12252.
U’Ren JM, Riddle JM, Monacell JT, Carbone I, Miadlikowska J, Arnold AE. Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi. Molecular ecology resources. 2014 Sep;14(5):1032–48.
U’Ren, Jana M., et al. “Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi.Molecular Ecology Resources, vol. 14, no. 5, Sept. 2014, pp. 1032–48. Epmc, doi:10.1111/1755-0998.12252.
U’Ren JM, Riddle JM, Monacell JT, Carbone I, Miadlikowska J, Arnold AE. Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi. Molecular ecology resources. 2014 Sep;14(5):1032–1048.
Journal cover image

Published In

Molecular ecology resources

DOI

EISSN

1755-0998

ISSN

1755-098X

Publication Date

September 2014

Volume

14

Issue

5

Start / End Page

1032 / 1048

Related Subject Headings

  • Specimen Handling
  • Sequence Analysis, DNA
  • Preservation, Biological
  • Plants
  • Molecular Sequence Data
  • Fungi
  • Evolutionary Biology
  • Endophytes
  • Diagnostic Errors
  • DNA Primers