Biology subjects

Hirvonen, T.

Publications and source records attributed to Hirvonen, T..

2 recordsLinked to original sources

bioRxiv2026

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

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 MGIs 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 (Spearmans {rho} = 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.

molecular biology↗

bioRxiv2025

Nicking Loop™: An infinite amplification of single-stranded circular DNA

Circular single-stranded DNA (CssDNA) offers unique advantages for molecular diagnostics and next-generation sequencing (NGS) due to its nuclease resistance and compatibility with rolling circle amplification. We present the Nicking Loop, a novel and versatile method for converting both single- and double-stranded DNA into CssDNA and enabling robust, unbiased amplification. This technique preserves the original DNA template composition and outperforms PCR in reproducibility and sensitivity, particularly for low variant allele frequencies. We demonstrate that Nicking Loop-amplified CssDNA is directly compatible with NGS platforms utilizing circular DNA as a sequencing template and introduce a novel loop-based indexing strategy that enables efficient sample multiplexing. Furthermore, we confirm the methods compatibility with nanopore sequencing technologies, highlighting its broad applicability across sequencing platforms. Our results establish Nicking Loop as an alternative to conventional linear library preparation, streamlining workflows while enhancing sequencing accuracy and efficiency. Beyond its immediate application in NGS, the method holds promise for broader use in molecular biology, e.g. in DNA data storage. This proof-of-concept study highlights the transformative potential of Nicking Loop in enabling a shift toward circular DNA libraries for new generation of assay preps and precision sequencing applications.

molecular biology↗