ApharSeq – extraction-free early-pooling protocol for massively multiplexed SARS-CoV-2 RNA sequencing

The global SARS-CoV-2 pandemic created a dire need for viral detection tests worldwide. Most current tests for SARS-CoV-2 are based on RNA extraction followed by quantitative reverse-transcription PCR assays. While automation and improved logistics increased the capacity of these tests, they cannot exceed the lower bound dictated by one extraction and one RT-PCR reaction per sample. Multiplexed next generation sequencing (NGS) assays provide a dramatic increase in throughput, and hold the promise of richer information including viral strains, host immune response, and multiple pathogens.

Here, researchers from the Hebrew University of Jerusalem establish a significant improvement of existing RNA-seq detection protocols. This workflow, ApharSeq, includes a fast and cheap RNA capture step, that is coupled to barcoding of individual samples, followed by sample-pooling prior to the reverse transcription, PCR and massively parallel sequencing. Thus, only one non-enzymatic step is performed before pooling hundreds of barcoded samples for subsequent steps and further analysis. The researchers characterize the quantitative aspects of the assay by applying ApharSeq to more than 500 clinical samples in a robotic workflow. The assay results are linear, and the empirical limit of detection is found to be Ct 33 (roughly 1000 copies/ml). A single ApharSeq test currently costs under 1.2$, and they estimate costs can further go down 3-10 fold. Similarly, the researchers estimate a labor reduction of 10-100 fold, automated liquid handling of 5-10 fold, and reagent requirement reduction of 20-1000 fold compared to existing testing methods.

ApharSeq overview


A) Barcoded and uniquely-identifiable RT primers are hybridized to samples in transport/lysis buffer or after a quick buffer replacement step. Paramagnetic beads are used to capture RNA and wash excess primers. B) Beads are pooled and RNA undergoes an RT/PCR reaction with pre-hybridized target-specific primers to generate a sequencing library. C) Libraries are sequenced and analysed, PCR duplicates are collapsed to molecular counts for detection and potentially more elaborate analyses, e.g. contact tracing by viral sequence analysis, host physiological state by mRNA analysis.

Chappleboim A, Joseph-Strauss D, Rahat A et al. (2020) ApharSeq: An Extraction-free Early-Pooling Protocol for Massively Multiplexed SARS-CoV-2 Detection. medRXiv [online preprint]. [abstract]

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