Error trade-offs in OTU assembly optimization.

<p>A. Total error (left ordinate) for <i>de novo</i> assemblies of <i>cpn</i>60 UT sequence reads from a synthetic community of 20 cloned targets, using a minimum identity value of 92% and a range of minimum overlap lengths (50–400 nucleotides). Raw total error (blue line), as well as error remaining after post-assembly primer trimming and clustering (red line), and after chimera removal (green line). Light blue bars indicate the percent of sequence reads identified as singletons in each assembly (right ordinate). B. Number of OTU assembled at each minimum overlap length. Each coloured segment of the stacked bar indicates a different member of the panel of 20 community members. The total number of OTU assembled is indicated on the top of each stack.</p

Sequence diversity across the 16S rRNA gene and <i>cpn</i>60 UT.

<p>Median percent identity of each of sequence to its nearest neighbour among the 16S rRNA <i>cpn</i>60 UT sequences from 1,394 bacterial genomes. Median percent identity was calculated for each 120 bp window along the length of the targets and identity values are plotted for the midpoint of each window. Due to target length variation, particularly among 16S rRNA genes, data is shown for windows for which at least 95% of the genomes could be included.</p

Definition of barcode regions based on established PCR primers.

<p>Definition of barcode regions based on established PCR primers.</p

Barcode gap analysis for 16S rRNA and <i>cpn</i>60 targets.

1<p>Median length of the target region, between amplification primer annealing sites.</p>2<p>Barcode gap is the difference between the median inter-specific distance and median intra-specific distance.</p>3<p>Distance is expressed in terms of substitutions/site.</p

Barcode gaps for candidate targets.

<p>Barcode gap analysis of potential barcodes derived from the 16S rRNA and <i>cpn</i>60 genes. Each panel shows the distribution of inter- (red) and intra-specific (green) distances in terms of percent of the total number of comparisons made (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0049755#pone-0049755-t002" target="_blank">Table 2</a>). In cases where percent values exceed 20, the actual value is indicated above an arrow on the relevant bar in the chart. For both V6-alternate and <i>cpn</i>60 UT, only distances up to 1.00 are plotted.</p

Sensitivity and specificity of the <i>C</i>. <i>purpurea cpn60</i>-targeted LAMP assay compared to visual rating (gold standard).

<p>Sensitivity and specificity of the <i>C</i>. <i>purpurea cpn60</i>-targeted LAMP assay compared to visual rating (gold standard).</p

Quantitative molecular diagnostic assays of grain washes for <i>Claviceps purpurea</i> are correlated with visual determinations of ergot contamination

<div><p>We examined the epiphytic microbiome of cereal grain using the universal barcode chaperonin-60 (<i>cpn60</i>). Microbial community profiling of seed washes containing DNA extracts prepared from field-grown cereal grain detected sequences from a fungus identified only to Class Sordariomycetes. To identify the fungal sequence and to improve the reference database, we determined <i>cpn60</i> sequences from field-collected and reference strains of the ergot fungus, <i>Claviceps purpurea</i>. These data allowed us to identify this fungal sequence as deriving from <i>C</i>. <i>purpurea</i>, and suggested that <i>C</i>. <i>purpurea</i> DNA is readily detectable on agricultural commodities, including those for which ergot was not identified as a grading factor. To get a sense of the prevalence and level of <i>C</i>. <i>purpurea</i> DNA in cereal grains, we developed a quantitative PCR assay based on the fungal internal transcribed spacer (ITS) and applied it to 137 samples from the 2014 crop year. The amount of <i>Claviceps</i> DNA quantified correlated strongly with the proportion of ergot sclerotia identified in each grain lot, although there was evidence that non-target organisms were responsible for some false positives with the ITS-based assay. We therefore developed a <i>cpn60</i>-targeted loop-mediated isothermal amplification assay and applied it to the same grain wash samples. The time to positive displayed a significant, inverse correlation to ergot levels determined by visual ratings. These results indicate that both laboratory-based and field-adaptable molecular diagnostic assays can be used to detect and quantify pathogen load in bulk commodities using cereal grain washes.</p></div

Ergot sclerotia observed in sample 9129 (Rye).

<p>Sclerotia are indicated by arrows. This sample had an ergot severity rating of 0.294% on a percentage weight basis (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173495#pone.0173495.t002" target="_blank">Table 2</a>). Scale bar indicates 1 cm.</p

<i>cpn60</i>-targeted LAMP assay linearity assessed by expressing T_p related to <i>C</i>. <i>purpurea</i> genome copies using the two LAMP detection systems evaluated in this study.

<p>The equations for each curve are: y = -9.03x+96.98 (calcein detection) and y = -1.90x+19.44 (isothermal detection). The correlation coefficients (r²) are 0.99 (calcein detection) and 0.95 (isothermal detection).</p

Phylogenetic analysis of <i>cpn60</i> UT sequences derived from microbial profiling and ergot sclerotia compared to reference sequences.

<p>Sequences are prefixed by cpnDB ID number (<a href="http://www.cpndb.ca" target="_blank">www.cpndb.ca</a>) and GenBank accession numbers (<a href="http://www.ncbi.nlm.nih.gov" target="_blank">www.ncbi.nlm.nih.gov</a>) are provided in parentheses where available. The tree was calculated using the Maximum Likelihood method based on the Tamura-Nei model [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173495#pone.0173495.ref022" target="_blank">22</a>] using MEGA6 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0173495#pone.0173495.ref023" target="_blank">23</a>]. The tree was bootstrapped (100 iterations) and numbers next to the nodes indicate the percentage of trees in which the associated taxa clustered together. Branch lengths correspond to the number of substitutions per site.</p