(B) Paired-primer approaches typically generate short amplicons flanked by upstream and downstream primers that are PCR amplified in non-overlapping pools. (C) Tiled-ClickSeq uses a single pool of primers at the reverse-transcription step with the upstream site generated by stochastic termination by azido-nucleotides. (D) 3’-Azido-blocked single-stranded cDNA fragments are ‘click-ligated’ using copper-catalyzed azide alkyne cycloaddition (CuAAC) to hexynyl functionalized Illumina i5 sequencing adaptors. Triazole-linked ssDNA is PCR amplified to generate a final cDNA library. (E) The structure of the final cDNA is illustrated indicating the presence of the i5 and i7 adaptors, the 12 N unique molecular identifier (UMI), the expected location of the triazole linkage, and the origins of the cDNA in the reads including the tiled primer-derived DNA, which is captured using paired-end sequencing. (F) The hypothetical read coverage over a viral genome is indicated in red, yielding overlapping ‘saw-tooth’ patterns of sequencing coverage. Longer fragment lengths with more extensive overlapping can be obtained using decreased AzNTP:dNTP ratios. (G) Final cDNA libraries are analyzed and size-selected by gel electrophoresis (2 % agarose gel). Duplicates of libraries synthesized from 8, 80, and 800 ng of input SARS-CoV-2 RNA input are shown. (H) Flowchart of the data processing and bioinformatic pipeline. Input data is in Blue, output data are in Green, scripts/processes are Purple.