DOI: 10.5061/dryad.8271f
title: Data from: Tissue storage and primer selection influence pyrosequencing-based inferences of diversity and community composition of endolichenic and endophytic fungi

description: 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 ca. 15-fold in a model data set. Third, we
 show that inferences regarding endolichenic diversity can be improved by
 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 artifacts.
description: ITS1 reads after quality
 trimmingFasta file containing
 all of the trimmed sequences (n=188,300) used in this study. Poor quality
 sequences, mycobiont sequences, and chimeric sequences have been removed.
 Singleton and doubleton OTU sequences are still
 present.pyro_ITS1.140bp.dist2dist3.slp.redundant.NoPelt.Chimeras.fastaITS1 read after quality trimming-group membershipThis file contains a list of all 188,300 sequences used in the study and their associated group membership. For example, samples in the study came from 3 host species (Pine, Pelt, Moss) x 3 individuals for each host (1,2,3) x 3 tissue storage methods (Fresh, CTAB, Silica). So the name Pine3CTAB denotes Pinus host # 3, tissue stored in CTAB buffer prior to DNA extraction.pyro.ITS1.140bp.good.NoPelt.Chimera.groupsOTU abundance matrix by host/microsite/treatmentThis file is the output from the mothur command make.shared for all 188,300 sequences in the study. It is an abundance matrix with the number of sequences for each sample at every level of sequence divergence (e.g., unique, 0.00, 0.01, 0.05). Level of 0.05 was used for the analyses for this study. This file contains all OTU.pyro_ITS1.140bp.dist2dist3.an.NoPelt.Chimera.shared