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

Area of Science:

• Genomics
• Molecular Biology
• Bioinformatics

Background:

• Standard 3' single-cell RNA-seq (scRNA-seq) uses poly(dT) primers, creating challenges in sequencing homopolymer regions.
• Historically, DNA sequencing errors at these priming sites limited accurate analysis of polyadenylation site usage.

Purpose of the Study:

• To evaluate Element Biosciences' avidity-based DNA sequencing for accurate homopolymer traversal.
• To assess the impact of sequencing through poly(T) sites on read alignment and polyadenylation site quantification in scRNA-seq.
• To compare Element's platform with Illumina sequencing for scRNA-seq applications.

Main Methods:

• Utilized Element Aviti instrument for DNA sequencing of scRNA-seq libraries.
• Performed paired-end sequencing and analyzed read alignments.
• Developed and applied an adjusted adapter trimming and alignment workflow for Element and Illumina data.
• Quantified polyadenylation site usage.

Main Results:

• Element Aviti accurately sequences through thymine homopolymers without compromising accuracy.
• Paired-end alignments facilitate direct polyadenylation site assignment, bypassing conventional method limitations.
• Improved alignment rates observed with an adjusted workflow, especially for longer reads.
• Element avidity sequencing demonstrates comparable performance to Illumina for standard scRNA-seq, particularly for polyadenylation site analysis.

Conclusions:

• Element avidity sequencing is a reliable alternative for scRNA-seq, especially for polyadenylation site analysis.
• The technology enables more direct and accurate assessment of polyadenylation events.
• Further investigation into emerging sequencing platforms for scRNA-seq is warranted.