Additional file 1 of A single workflow for multi-species blood transcriptomics

Supplementary Material 1

Longitudinal assessment of K103N mutants frequency by allele-specific PCR during the intermittent off-therapy periods.

<p>w, week; Y, yes; N, no; *, K103N mutants also detected by direct sequencing; dashes, samples not available.</p

Correlation between K103N mutants quantified by allele-specific real-time PCR and ultra-deep pyrosequencing.

<p>A. Frequencies of K103N mutants. B. Number of K103N mutant copies The correlation was estimated by calculating Spearman's rank correlation coefficient for 16 samples successfully quantified by both methods for the AAC and/or AAT mutated codons.</p

K103N mutants in 11 patients quantified by allele-specific real-time PCR and ultra-deep pyrosequencing.

<p>The frequencies of K103N mutants are shown with corresponding absolute numbers of K103N log₁₀ copies per mL of plasma in brackets.</p><p>w, week; AAC and AAT codons encode the asparagine « N » at position 103; dashes, samples not available.</p

Sensitivity of the allele-specific PCR assay for detecting minor K103N mutants and reciprocal validation with ultra-deep pyrosequencing.

<p><b>A</b>. Plot of the measured frequencies of K103N mutants versus the input template, assessed on four independent experiments of mixtures of wild-type and K103N mutants at various frequencies. The inter-assay variability is shown by error bars representing the standard deviation. The assay detects K103N mutants down to a frequency of 0.01% in all experiments. <b>B</b>. Measured frequencies of K103N mutants on mixtures of wild-type and K103N mutants with known proportions of K103N. <b>C</b>. Correlation between measurements by the two methods.</p

Higher efavirenz half-life in patients in whom K103N emerged than in whom it did not.

<p>The Wilcoxon rank sum test was used for the comparison.</p

Detection threshold of ultra-deep pyrosequencing for detecting minor K103N variants in function of the read number.

<p>The mean error rate of pyrosequencing at codon 103 was 0.0047 [CI99, 0.00085–0.00856]. The upper confidence limit of the error rate was used to calculate the sensitivity of pyrosequencing for a given number of reads. Poisson distribution was used to distinguish authentic variants from artefactual sequences resulting from errors arising during PCR amplification and ultra-deep pyrosequencing. Only those variants whose frequency of occurrence yielded a <i>P</i> value of <0.001 according to the Poisson model were considered authentic.</p