Robust Multichannel Encoding for Highly Multiplexed Quantitative PCR

Abstract

The gold standard of molecular pathogen detection is the quantitative polymerase chain reaction (qPCR). Modern qPCR instruments are capable of detecting 4−6 analytes in a single sample: one per optical detection channel. However, many clinical applications require multiplexing beyond this traditional single-well capacity, including the task of simultaneously testing for SARS-CoV-2 and other respiratory pathogens. This can be addressed by dividing a sample across multiple wells, or using technologies such as genomic sequencing and spatial arrays, but at the expense of significantly higher cost and lower throughput compared with single-well qPCR. These trade-offs represent unacceptable compromises in high-throughput screening scenarios such as SARS-CoV-2 testing. We demonstrate a novel method of detecting up to 20 targets per well with standard qPCR instrumentation: high-definition PCR (HDPCR). HDPCR combines TaqMan chemistry and familiar workflows with robust encoding to enable far higher levels of multiplexing on a traditional qPCR system without an increase in cost or reduction in throughput. We utilize HDPCR with a custom 20-Plex assay, an 8-Plex assay using unmodified predesigned single-plex assays from Integrated DNA Technologies and a 9-Plex pathogen panel inclusive of SARS-CoV-2 and other common respiratory viruses. All three assays were successful when tested on a variety of samples, with overall sample accuracies of 98.8, 98.3, and 100%, respectively. The HDPCR technology enables the large install base of qPCR instrumentation to perform mid-density multiplex diagnostics without modification to instrumentation or workflow, meeting the urgent need for increased diagnostic yield at an affordable price without sacrificing assay performance.

Multiplexed CRISPR-based microfluidic platform for clinical testing of respiratory viruses and identification of SARS-CoV-2 variants

“…As examples, reverse transcriptionquantitative PCR (RT-qPCR) is high-throughput by testing at least 88 samples, but for 1-3 analytes at a time; multiplexed techniques such as Cepheid Xpert Xpress can detect 4 respiratory viruses in up to 16 samples per run, and BioFire can detect 22 respiratory pathogens in 1 sample simultaneously 13 . Only a few clinical diagnostic methods comprehensively detect SARS-CoV-2 variant mutations [14][15][16] which is why this has largely been achieved through nextgeneration sequencing (NGS) 17,18 , even though it is time-consuming, expensive, and requires bioinformatic expertise to interpret 6,[19][20][21][22] .…”

An adaptive filtering framework for non-specific and inefficient reactions in multiplex digital PCR based on sigmoidal trends

“…final fluorescence intensity or FFI) and PCR platforms with multiple fluorescent channels. 17 On the other hand, Moniri et al in 2020 proposed a new approach called Amplification Curve Analysis (ACA) for single channel multiplexing without explicitly extracting features. The ACA method comprises a supervised ML classifier to analyze kinetic information encoded in the entire amplification curve, by looking into sigmoidal shapes across different targets.…”

Virtual-Partition Digital PCR for High-Precision Chromosomal Counting Applications

“…11−15 However, this difficulty can be overcome by applying high-definition PCR (HDPCR). 16 This technique was originally developed for qPCR applications, where it allows for multiplexing of multiple targets per color channel by generating a distinct end-point intensity for each possible combination of targets present. In this paper, we leverage HDPCR in a dPCR application, generating multiple distinct droplet amplitude clusters per color channel.…”

“…One way around this partitioning variance limitation is to apply enhanced assay design to generate multiple distinct signal clusters corresponding to different combinations of target regions in the partitions. Historically, this process has proven difficult, limiting such multiplexing applications to two or three targets per optical color channel. − However, this difficulty can be overcome by applying high-definition PCR (HDPCR) . This technique was originally developed for qPCR applications, where it allows for multiplexing of multiple targets per color channel by generating a distinct end-point intensity for each possible combination of targets present.…”