Improved characterization of 3' single-cell RNA-seq libraries with paired-end avidity sequencing.

Prevailing poly(dT)-primed 3' single-cell RNA-seq protocols generate barcoded cDNA fragments containing the reverse transcriptase priming site or in principle the polyadenylation site. Direct sequencing across this site was historically difficult because of DNA sequencing errors induced by the homopolymeric primer at the 'barcode' end. Here, we evaluate the capability of 'avidity base chemistry' DNA sequencing from Element Biosciences to sequence through the primer and enable accurate paired-end read alignment and precise quantification of polyadenylation sites. We find that the Element Aviti instrument sequences through the thymine homopolymer into the subsequent cDNA sequence without detectable loss of accuracy. The additional sequence enables direct and independent assignment of reads to polyadenylation sites, which bypasses the complexities and limitations of conventional approaches but does not consistently improve read mapping rates compared to single-end alignment. We also characterize low-level artifacts and demonstrate necessary adjustments to adapter trimming and sequence alignment regardless of platform, particularly in the context of extended read lengths. Our analyses confirm that Element avidity sequencing is an effective alternative to Illumina sequencing for standard single-cell RNA-seq, particularly for polyadenylation site measurement but do not rule out the potential for similar performance from other emerging platforms.