Classifiability of 16S sequence data is differentially impacted by sequencing technology, taxonomic family and body region.

<p>For each of the HMP body regions, the relationship between the average frequency of a given bacterial family (y-axis) versus the contribution of these families to the unclassifiability issue (x-axis) is plotted for (B) 3730 and (C) 454. Only window V3–V5 is presented in 454 results. Classification was performed on quality- and chimera-filtered sequences and classifications assigned only if the RDP Classifier result had a confidence score above 80%.</p

Evaluation of 16S rDNA-Based Community Profiling for Human Microbiome Research

<div><p>The Human Microbiome Project will establish a reference data set for analysis of the microbiome of healthy adults by surveying multiple body sites from 300 people and generating data from over 12,000 samples. To characterize these samples, the participating sequencing centers evaluated and adopted 16S rDNA community profiling protocols for ABI 3730 and 454 FLX Titanium sequencing. In the course of establishing protocols, we examined the performance and error characteristics of each technology, and the relationship of sequence error to the utility of 16S rDNA regions for classification- and OTU-based analysis of community structure. The data production protocols used for this work are those used by the participating centers to produce 16S rDNA sequence for the Human Microbiome Project. Thus, these results can be informative for interpreting the large body of clinical 16S rDNA data produced for this project.</p> </div

454 sequences have a higher error rate, mainly resulting from an increased insertion and deletion rate.

<p>(A) For all the quality and chimera filtered 3730 and 454 sequences generated for the MC sample, an alignment-based estimation of errors, including insertions, deletions, and substitutions was performed. For each of the different sequencing approaches, the cumulative frequency distribution of the percent error per sequence is shown for assembled 3730 sequences generated with short capillaries (green), long capillaries (red), and three reads per clone (yellow), and 454 reads spanning the variable regions V1–V3 (light blue), V3–V5 (dark blue), and V6–V9 (fuchsia). A vertical line at 1% was added as a visual aid for upper limit of an acceptable error threshold. (B) Boxplots show the average percentage of errors per read, per sequence approach and per error type, including substitutions, insertions, and deletions. Outliers are not shown.</p

Deviation from expected in the 16S based Mock Community member representation can partially be explained by primer mismatch, not by %GC differences.

<p>The 20 bacterial organisms of the Mock Community are represented by corresponding genus (n = 18) along the bottom of the figure, and across the four panels (DNA from <i>Candida albicans</i> was included in this mock community, but not shown here). (A) The distribution of reads over the 18 genera; The expected frequencies (grey) in the community determined by whole genome shotgun (WGS) sequencing and classified by mapping to reference genomes using BWA, and the observed frequencies determined by 454 reads (red) or 3730 sequences (blue), classified by BLASTn. Error bars indicate standard error from technical replicates. (B) Deviations from expected frequencies as calculated by subtracting expected % from the observed. (C) The average %GC is shown for all its 16S genes, and for their whole genomes. (d) The lowest percent mismatch between primer used in production protocols (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039315#pone.0039315.s001" target="_blank">Protocols S1</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0039315#pone.0039315.s002" target="_blank">S2</a>) and any 16S gene copy is shown for each organism; primers are grouped by sequencing technology and 16S window.</p

Overview amplicons and reads generated for both the 3730 and 454 sequencing.

<p>On a schematic representation of the 16S rDNA gene, the known variable regions and the primers used in this study are indicated. Positions and numbering are based on the <i>Escherichia coli</i> reference sequence. The amplicons generated by each primer set are marked in red, and sequencing directions and expected lengths are indicated in orange for 3730 and green for 454.</p

The <i>Lachnospiraceae</i> 16S diversity observed in stool samples is greater than from known reference resources.

<p>A phylogenetic tree constructed with 16S sequences from RDP’s training set (light blue, n = 34), publicly available genomes from human isolates (green, n = 26), publicly available HMP genomes (dark blue, n = 44), and sequences from aggregate stool samples that could be classified at the genus level (dark grey, n = 63) and that remained unclassified at the genus level (light grey, n = 408).</p

Illustration of how flawed taxonomic schemes and sequence quality can result in incorrect classifications.

<p>The phylogenetic trees were created starting from the full-length reference sequences that were used to train RDP’s taxonomic scheme version 5 for <i>Pseudomonas</i> and <i>Azomonas</i> (A), and <i>Neisseriaceae</i> (B), respectively. These sequences were clustered into 3% OTUs with mothur and representatives for each OTU were selected for building a tree with FastTree. The number of sequences belonging to each OTU is indicated in brackets.(C) Scatter density plots of percent low quality (QV<20) bases per read versus read length is shown for the misclassified reads (red) compared to their correctly classified counterparts (blue).</p

Error by position profiles indicate hotspots for error.

<p>To visualize where sequencing errors were concentrated along the length of the 16S sequence for each sequencing technology, a root mean square deviation (RMSD) plot was generated for (A) 3730 sequence and (B) 454 read data. The RMSD plot is a graphical representation of the differences in nucleotide distribution between a reference sequence and the samples of interest, for each position along the length of the reference. This figure shows the results for <i>Neisseria meningitidis</i> specifically, but is representative of the profiles observed for the other strains in the MC.</p

Chimera rates in 16S data sets.

*<p>Values are averages ± STDEV calculated from multiple replicates of MC, and from replicates of multiple clinical samples originating from different body sites.</p