Application of the Nicking Loop™ targeted library preparation method to DNBSEQ™ sequencing

ABSTRACT Nicking Loop™ is a PCR-free targeted library preparation method that combines conversion of linear target DNA into circular single-stranded DNA (CssDNA) library with early sample indexing in a single step. The resulting CssDNA libraries can be either directly sequenced or optionally amplified, offering maximum flexibility across sequencing applications. This study demonstrates the compatibility of Nicking Loop™ circular libraries with a MGI’s DNBSEQ™ platform. Compatibility was evaluated against established linear Nicking Loop™ libraries sequenced on Illumina MiSeq platform. Using synthetic reference samples with defined variant allele frequencies, Nicking Loop™ method demonstrated matching performance across both library formats and sequencing platforms. Key quality metrics, including unique molecular identifier (UMI) distributions, error profiles and VAF detection, were all highly consistent. Both library types generated over 97% singleton UMIs, indicating uniform template sampling, and VAF measurements were strongly concordant across platforms (Spearman’s ρ = 0.939). Collectively, these findings demonstrate that Nicking Loop™ method is directly applicable to circular NGS platforms, such as DNBSEQ™, strongly supporting its use as a platform-agnostic library preparation strategy for targeted sequencing applications.