Supporting Information Table S1 and Methods S1 Methods S1 Power analysis for multivariate data. Recent developments in computational biology have made it possible to conduct power analyses on multivariate datasets (La Rosa et al., 2012, 2013). Specifically the R-package HMP (La Rosa et al., 2015) was used to examine changes in experimental power with changes in the number of samples per treatment and sequences per sample. We used two published arbuscular mycorrhizal fungi (AMF) community datasets Denmark (Lekberg et al., 2012) and Montana (Lekberg et al., 2013) for which data consisted of multiple samples per site, and samples were in the form of sequence abundance. Data frames were set up such that each row was a single sample and each column a single taxon, with the final column labelled rare taxa in which all taxa with an experiment-wide sequence abundance <1% of the total number of sequences were combined (La Rosa et al., 2012). All subsequent analyses were performed with raw count data using the DM.MoM function to fit the recommended Dirichlet-multinomial (DM) model. For an experimental significance level of α = 0.05 we calculated the experimental power for varying numbers of sequences and varying numbers of samples using the function mc.xmcupo for each of the two datasets (see Table 1). Additionally the effect size function Xmcupo.effectsize and Generalized Wald-type test function Xmcupo.sevsample were applied to the Denmark and Montana datasets examine whether the original conclusions about community differences were supported using the HMP approach. In each case analysis with HMP supported the original conclusions.
Table S1 Published most frequently used for arbuscular mycorrhizal fungi (AMF) studies Reference Primer set Target region White et al. (1990) NS1, NS2, NS4, NS5, NS8 SSU ITS1, ITS4 ITS Notes Universal eukaryote used as first PCR and followed by a nested specific PCR, or in combination with specific Simon et al. (1992) NS31 SSU Universal eukaryote primer vantuinen et al. (1998) LR1, NDL22 LSU Universal eukaryote Helgason et al. (1998) AM1 SSU Paired with universal forward primer, can amplify other groups of fungi, limited coverage of Paraglomeraceae Trouvelot et al. (1999) FLR2 LSU Paired with LR1 for fungi only Kjøller & Rosendahl (2000) LSURK4f, LSURK7r LSU Glomus group primer, nested within LR1-NDL22 Redecker et al. (2000) ARCH1311, LETC1670, GLOM1310, ACAU1660, GLOM5.8R, GIGA5.8R GeoA1, GeoA2, Geo10, Geo11, GeoNS1, ART4 SSU & ITS Taxon group specific Schwarzott & Schüssler (2001) SSU AMF specific Renker et al. (2003) SSUGlom1, LSUGlom1 ITS Primary PCR with restriction digest prior to secondary amplification using universal ITS Gollotte et al. (2004) FLR3, FLR4 LSU AMF specific, nested within LR1-FLR2 Saito et al. (2004) AMV4.5F, AMV4.5R, SSU AMF specific
AMV4.5NF, AMV4.5NR Sato et al. (2005) AMDGR SSU AMF specific reverse primer with improved coverage for basal lineages Wubet et al. (2006) GlomerWT0, GlomerWT1, GlomerWT2,GlomerWT3, GlomerWT4, Glomer1536 SSU General AMF specific and group specific Santos-Gonzalez et al. (2007) AM2, AM3 SSU Variants of AM1 that increase taxon coverage Lee et al. (2008) AML1, AML2 SSU Longer fragment than NS31-AM1, improved AMF taxon coverage, amplifies some plants Krüger et al. (2009) SSUmAf, SSUmCf, LSUmBr, LSUmAr SSU- ITS-LSU Composite primer mixtures for high taxon coverage Lekberg et al. (2012) Glo454 LSU Combined with NDL22 for 454 sequencing References where original primer sequence was published are cited, with notes on coverage and typical primer combinations. SSU, small subunit; ITS, internal transcribed spacer; LSU, large subunit.
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