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Increasing ecological inference from high throughput sequencing of fungi in the environment through a tagging approach
Author(s) -
TAYLOR D. LEE,
BOOTH MICHAEL G.,
MCFARLAND JACK W.,
HERRIOTT IAN C.,
LEN NIALL J.,
NUSBAUM CHAD,
MARR THOMAS G.
Publication year - 2008
Publication title -
molecular ecology resources
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.96
H-Index - 136
eISSN - 1755-0998
pISSN - 1755-098X
DOI - 10.1111/j.1755-0998.2008.02094.x
Subject(s) - biology , unifrac , phylogenetic tree , dna sequencing , genetics , ascomycota , computational biology , library , internal transcribed spacer , amplicon , ribosomal dna , evolutionary biology , polymerase chain reaction , gene , 16s ribosomal rna
High throughput sequencing methods are widely used in analyses of microbial diversity, but are generally applied to small numbers of samples, which precludes characterization of patterns of microbial diversity across space and time. We have designed a primer‐tagging approach that allows pooling and subsequent sorting of numerous samples, which is directed to amplification of a region spanning the nuclear ribosomal internal transcribed spacers and partial large subunit from fungi in environmental samples. To test the method for phylogenetic biases, we constructed a controlled mixture of four taxa representing the Chytridiomycota, Zygomycota, Ascomycota and Basidiomycota. Following cloning and colony restriction fragment length polymorphism analysis, we found no significant difference in representation in 19 of the 23 tested primers. We also generated a clone library from two soil DNA extracts using two primers for each extract and compared 456 clone sequences. Community diversity statistics and contingency table tests applied to counts of operational taxonomic units revealed that the two DNA extracts differed significantly, while the pairs of tagged primers from each extract were indistinguishable. Similar results were obtained using UniFrac phylogenetic comparisons. Together, these results suggest that the pig‐tagged primers can be used to increase ecological inference in high throughput sequencing projects on fungi.