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Data is normalized to the most dilute sample to be represented on the same scale.
The detection limits, calculated as 3 × SD where SD is the standard deviation of the most dilute sample, were of the order of a few μg mL−1.
Although some contaminating gDNA remained after column purifications, -RT controls for each gene were more than six cycles greater than the most dilute sample for that gene and thus would have a minimal effect on quantification (Laurell et al. 2012).
As Figure 5 also shows, even in the most dilute sample that contains only 0.5% WBC, 3 ± 1.5 Chlamydia positive cells above background were detectible in the 10,000 cell sample.
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In repeated runs of this dilution series throughout the experiment, however, the most dilute samples (10 copies (5 μl -1) occasionally failed to amplify, presumably due to the stochasticity of PCR reactions with low template.
Calculating the Shannon-Entropy for 4-mer distributions from all metagenomes confirmed that the two most dilute samples suffered from a particularly biased raw sequence read composition.
The most concentrated vector dilutions resulted in near 100% transduction, whereas the most diluted samples resulted in TEs near zero (* in Figure 2) and were omitted from the linear regression.
To specifically examine INF-A subtype detection, we tested serially diluted irradiated whole extracts and cell culture supernatants of reference strains of H1N1, H3N2, and Indonesian and Vietnamese H5N1 strains with Influenza Profile 1 (Table 1), with the most diluted samples containing only 1-10 cofies of RNA virus/μl.
All NTC did not indicate the melt temperature of the GOI amplicon, and 5 of 6 investigated genes had more than 7 Ct between the average NTC Ct and the most dilute unknown sample (troponin I, slow skeletal muscle was only 3.8 Ct from the average NTC primer dimer; SABiosciences).
The deviation of mean equilibration temperatures of the original reservoir fluid compared to the sample composition is very low, reaching maximum ~15 K for sample Liu, which is the most highly diluted sample (1 1).
With respect to the serially diluted synthetic templates, the two most diluted calibration spikes (cYIR09 and cYIR10) in the samples with 1000 cells were below the threshold, while for 250 cell samples, the five most diluted calibration spikes cYIR06-cYIR100) were below threshold.
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