Pyrosequencing of 28S rRNA gene amplicons
28S rRNA gene was applied to investigate fungal taxonomy in this study.
After combining with a single sample-specific 8-mer tag sequence
separated by a 2-bp spacer sequence, the primers LR0R
(5’-ACCCGCTGAACTTAAGC-3’) and LR3 (5’-CCGTGTTTCAAGACGGG-3’) were applied
to amplify the D1-D2 region of 28S rRNA gene, which generated a gene
amplicon of about 650 bp. The 100
µL PCR system was composed of 50 mM KCl, 10 mM Tris-HCl at pH of 8.3,
0.1% Triton X-100, 2.5 mM MgCl2, 200 µM dNTPs
(Invitrogen Life Technologies, Carlsbad, CA, USA), 5 units of Taqpolymerase, 0.1 µg BSA (New England Biolabs Inc., Ipswich, MA, USA), 0.4
µM each primer and 12.5 ng DNA template. PCR thermocycling conditions
were as follows: 3 min at 94 °C for initialization, followed by 35
cycles of 1 min at 94 °C for denaturation, 45 secs at 53 °C for
annealing, 1 min at 72 °C for
extension, and finally 15 min at 72 °C before maintained at 4 °C. For
each sample, five to ten PCR reactions were performed. PCR products were
combined and purified by agarose gel electrophoresis, and then collected
with QIAquick Gel Extraction Kit (Qiagen, Venlo, Netherlands). PCR
products were quantified by PicoGreen using a FLUOstar Optima (BMG
Labtech, Jena, Germany). Equimolar uniquely-tagged amplicons from each
sample were then combined into one library.
Tags
separated raw sequences via the function “Split by tag” of the IEG
Galaxy pipeline (http://zhoulab5.rccc.ou.edu:8080). They were further
cleaned via LUCY based on the following thresholds: maximum average
error at 0.01, maximum error at the ends at 0.01, minimum score at 21,
window size at 50, minimum length at 200, and allowable ambiguous base
number (‘N’) at 10. Only sequences with no ‘N’ were accepted and were
clustered into operational taxonomic units (OTUs) using UCLUST at the
97% identity.
OTU singletons were removed and
OTUs annotation was performed by the RDP Classifier with the latest
Fungal LSU training set 11
(http://rdp.cme.msu.edu/classifier/classifier.jsp) at the 50%
confidence interval. We used raw
OTU abundance in the following data analyses.