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Five thousand lymphocyte events are acquired per sample with threshold for acquisition set on the FL1 (FITC) channel.
With the Panleucogate gating strategy (9, 10), 5,000 lymphocytes are acquired per sample with threshold for acquisition set on the forward scatter (size) channel.
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A stepwise regression was performed in each replicated sample with thresholds of p = 0.05 for variable selection and for variable elimination [ 19].
Samples with threshold cycle (C T) values below 40 were considered positive (28).
$$\end{document} Samples with a threshold cycle higher than the chosen cutoff point are classified as negative, whereas samples with threshold cycle lower than the cutoff point are classified as positive [ 30].
Samples with threshold cycle (Ct) values ≥ 35 or those where the standard deviation across a triplicate was ≥ 0.5 were considered failed reactions and re-run.
Test samples with threshold cycle numbers that fell outside the range defined by the standard curves were rerun at different concentrations to ensure that they were amplified within the linear range.
Any sample with a threshold cycle (CT) of <35 was considered to be positive for WNV.
People who refused to participate in the study could benefit from routine screening (one sample with positivity threshold at 100 ng ml−1).
Only samples with a threshold lower than 0.05 were used for sequencing library construction (Table S5).
Samples with a threshold crossing point >32 cycles for the genomic PCR were deemed poorly amplifiable and excluded from analysis.
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