Direct Amplification, Sequencing and Profiling of <i>Chlamydia trachomatis</i> Strains in Single and Mixed Infection Clinical Samples

<div><p>Sequencing bacterial genomes from DNA isolated directly from clinical samples offers the promise of rapid and precise acquisition of informative genetic information. In the case of <i>Chlamydia trachomatis</i>, direct sequencing is particularly desirable because it obviates the requirement for culture in mammalian cells, saving time, cost and the possibility of missing low abundance strains. In this proof of concept study, we developed methodology that would allow genome-scale direct sequencing, using a multiplexed microdroplet PCR enrichment technology to amplify a 100 kb region of the <i>C. trachomatis</i> genome with 500 1.1–1.3 kb overlapping amplicons (5-fold amplicon redundancy). We integrated comparative genomic data into a pipeline to preferentially select conserved sites for amplicon design. The 100 kb target region could be amplified from clinical samples, including remnants from diagnostics tests, originating from the cervix, urethra and urine, For rapid analysis of these data, we developed a framework for whole-genome based genotyping called <i>binstrain</i>. We used <i>binstrain</i> to estimate the proportion of SNPs originating from 14 <i>C. trachomatis</i> reference serotype genomes in each sample. Direct DNA sequencing methods such as the one described here may have an important role in understanding the biology of <i>C. trachomatis</i> mixed infections and the natural genetic variation of the species within clinically relevant ecological niches.</p></div

Phylogeny of 100

<p>Tree calculated in the same manner as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099290#pone-0099290-g002" target="_blank">Figure 2</a> but instead based on the 100 kb region of the <i>C. trachomatis</i> genome selected for targeted amplification. The colors are the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099290#pone-0099290-g003" target="_blank">Figure 3</a>.</p

Whole-genome phylogeny of the reference <i>C. trachomatis</i> strains used in this study.

<p>The tree was constructed using a neighbor-joining algorithm based on whole-genome alignment. All the branches were supported by 100% confidence in 100 bootstrap sampling except for the A/HAR-13/B/Jali20 branch. All nodes with bootstrap support <100% are designated with an asterisk. Each leaf and internal branch has the number of SNPs unique to this branch compared to the CT-ASR, reconstructed ancestral sequence. The leaves are colored by membership of major <i>C. trachomatis</i> Clades <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099290#pone.0099290-Joseph1" target="_blank">[7]</a>: yellow, blue, green and red for Clades 1–4, respectively.</p

The number of SNPs recovered through the RainDance targeted capture methodology for each of the single strain purified <i>C. trachomatis</i> samples used in this study that already has a genome sequence available.

<p>The true/expected SNPs were identified by performing a reference mapping of the sample reads to their corresponding genome sequence data.</p><p>*There is no complete Clinical D reference sequence.</p

Flow chart representing the workflow of the entire procedure of target amplification, sequencing and genotype profiling performed in this study.

<p>The dashed box represents the core analysis starting with downloaded reference genomes necessary for primer design and <i>binstrain</i> analysis. CT-ASR is the <i>C. trachomatis</i> ancestral reconstruction sequence.</p

Primer design strategy.

<p>Diverse <i>C. trachomatis</i> genomes were aligned using Whole Genome Alignment (WGA) software against the <i>C. trachomatis D/UW3/CX</i> reference sequence and SNPs (hash lines) were identified (a small number of indels were also identified but were omitted from the figure to make it simpler). The genome was divided into 100 bp blocks. Blocks with a threshold of two or more SNPs are labeled in black and correspond to gray regions in the genome). Starts and ends for primers (amplicon regions in dashed lines) were designed to avoid variable blocks (the black portions of the barcode). Primers were designed to allow approximately 5-fold overlapping amplicon coverage.</p

Summary of <i>binstrain</i> analysis of selected gDNA and clinical sample data.

<p>Format is the same as <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0099290#pone-0099290-g002" target="_blank">Figure 2</a>. Note that for a potential mixed infection, we would not be able to currently distinguish between multiple the presence of recombinant and non-recombinant strains, just the proportion of the genotype specific SNPs represented by the <b>β</b> values.</p

Details of the <i>C. trachomatis</i> samples along with the preliminary analysis results that were successfully amplified in this study.

1<p>Droplet PCR amplification failed.</p

Sample population distributions of concordant and discordant samples (A) and results of multiplex PCR and Amplicor assays versus sequencing to resolve discordant calls (B).

<p>Numbers noted on each bar represent the number of samples that were or were not confirmed by sequencing. Following resolution of discrepants by sequencing, 129 (76+53) and 88 (76+12) samples were positive by multiplex and Amplicor assays, respectively. An overall concordance rate of 82.9% was found between the multiplex assay and sequencing as compared to 17.1% for the Amplicor assay.</p

Representative clinical sample 9-plex profiles.

<p>Approximately half the positive electropherograms showed all 9 amplicons with saturated (Panel A) or intermediate (Panel B) peak signals, suggesting high copy numbers of <i>Ct</i>. The remaining positive samples generated incomplete profiles, having fewer than 9 amplicons and suggesting low <i>Ct</i> numbers. For example, Panel C shows the pCT peak and three other amplicons, Panel D shows pCT and two other amplicons, and Panel E shows no pCT and two other amplicons in blue and red. Panel F is a negative clinical sample showing nonspecific peaks (circled in green) that are readily distinguished from <i>Ct</i> amplicons based on molecular weight and fluorescent dye color. The 353 bp peak in blue is readily distinguished from a true 349 bp pCT amplicon in blue. The 230 bp peak in red is readily distinguished from the 4 red-labeled specific amplicons, all of which differ in size from the artifact by at least 120 bp.</p