Factors that affect large subunit ribosomal DNA amplicon sequencing studies of fungal communities: classification method, primer choice, and error.

PloS One
T M Porter, G Brian Golding

Abstract

Nuclear large subunit ribosomal DNA is widely used in fungal phylogenetics and to an increasing extent also amplicon-based environmental sequencing. The relatively short reads produced by next-generation sequencing, however, makes primer choice and sequence error important variables for obtaining accurate taxonomic classifications. In this simulation study we tested the performance of three classification methods: 1) a similarity-based method (BLAST + Metagenomic Analyzer, MEGAN); 2) a composition-based method (Ribosomal Database Project naïve bayesian classifier, NBC); and, 3) a phylogeny-based method (Statistical Assignment Package, SAP). We also tested the effects of sequence length, primer choice, and sequence error on classification accuracy and perceived community composition. Using a leave-one-out cross validation approach, results for classifications to the genus rank were as follows: BLAST + MEGAN had the lowest error rate and was particularly robust to sequence error; SAP accuracy was highest when long LSU query sequences were classified; and, NBC runs significantly faster than the other tested methods. All methods performed poorly with the shortest 50-100 bp sequences. Increasing simulated sequence error reduced clas...Continue Reading

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Citations

Sep 10, 2013·Applied and Environmental Microbiology·C Ryan PentonJames M Tiedje
Aug 1, 2015·The Journal of General and Applied Microbiology·Yoon-Jong NamJong-Guk Kim
Jul 1, 2015·Antonie van Leeuwenhoek·Manee TantirungkijSavitree Limtong
Jan 3, 2018·Molecular Ecology·Teresita M Porter, Mehrdad Hajibabaei
Oct 18, 2015·Applied and Environmental Microbiology·Jiarong GuoJames M Tiedje
Apr 18, 2019·Microbial Biotechnology·Susanne G LangerFrank R Bengelsdorf
Jun 26, 2020·Biochemistry and Molecular Biology Education : a Bimonthly Publication of the International Union of Biochemistry and Molecular Biology·Ivo R HornBarbara Gravendeel
Mar 29, 2013·The New Phytologist·Björn D LindahlHåvard Kauserud
Nov 13, 2012·PloS One·Anders LanzénTim Urich

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Methods Mentioned

BETA
PCR
amplicon-based sequencing

Software Mentioned

BLAST MEGAN
Metagenomic Analyzer parsing ( BLAST + MEGAN )
MEGAN
custom Perl scripts
BLAST
Bioconductor package ‘ genomes ’
Statistical Assignment Package ( SAP )
+ MEGAN
custom Perl script
R

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