Analysis of TaqMan Array Cards Data by an Assumption-Free Improvement of the maxRatio Algorithm Is More Accurate than the Cycle-Threshold Method

Abstract

Quantitative PCR diagnostic platforms are moving towards increased sample throughput, with instruments capable of carrying out thousands of reactions at once already in use. The need for a computational tool to reliably assist in the validation of the results is therefore compelling. In the present study, 328 residual clinical samples provided by the Public Health England at Addenbrooke's Hospital (Cambridge, UK) were processed by TaqMan Array Card assay, generating 15 744 reactions from 54 targets. The amplification data were analysed by the conventional cycle-threshold (CT) method and an improvement of the maxRatio (MR) algorithm developed to filter out the reactions with irregular amplification profiles. The reactions were also independently validated by three raters and a consensus was generated from their classification. The inter-rater agreement by Fleiss' kappa was 0.885; the agreement between either CT or MR with the raters gave Fleiss' kappa 0.884 and 0.902, respectively. Based on the consensus classification, the CT and MR methods achieved an assay accuracy of 0.979 and 0.987, respectively. These results suggested that the assumption-free MR algorithm was more reliable than the CT method, with clear advantages for the diagnostic settings.

Differences in epidemiology of enteropathogens in children pre- and post-rotavirus vaccine introduction in Kilifi, coastal Kenya

“…A custom TaqMan Array Card (TAC) spotted with lyophilized target-specific primers and probes was used to detect enteropathogens from the total NAE. The GASTRO v4.0K TAC was designed by MC at Public Health England (PHE), Cambridge, England [16] and manufactured by ThermoFisher, USA. GASTRO v4.0K screened for 28 enteropathogens: 12 viruses, 10 bacteria, 5 protozoa and 1 helminth (supplementary Table 1).…”

“…A Rotarix ® vaccine-specific assay targeting the NSP2 gene was included to identify RVA vaccine shedding due to prior vaccination. The molecular probes were labeled at the 5’ end with 6-carboxyfluorescein (FAM) reporter dye and NFQ-MGB quencher dye at the 3’ end [16].…”

maxRatio improves the detection of samples with abnormal amplification profiles on QIAgen’s artus HIV-1 qPCR assay

“…The FP method generated the Cq by registering the fractional cycle where the fluorescence passed the threshold of 0.2 units. The maxRatio transform of the amplification data and determination of the cut-offs were computed as previously described 9 , 10 . Different operators visually inspected the reaction’s profiles and classified each reaction as either passed or failed.…”

“…An alternative method, maxRatio, was introduced to overcome these issues 9 . It has been reported that maxRatio conferred a marginal increase in assay accuracy over FP 10 , 11 .…”

maxRatio improves the detection of samples with abnormal amplification profiles on QIAgen’s artus HIV-1 qPCR assay

“…An alternative method, maxRatio, was introduced to overcome these issues 9 . It has been reported that maxRatio conferred a marginal increase in assay accuracy over FP 10,11 . FP provides only a quantification cycle (Cq) value, which is then used to calculate VL.…”