BRIF-Seq: Bisulfite-Converted Randomly Integrated Fragments Sequencing at the Single-Cell Level

Area of Science:

• Epigenetics
• Genomics
• Plant Science

Background:

• Single-cell bisulfite sequencing (scBS-seq) faces challenges with read under-representation in highly methylated genomic regions.
• Illumina sequencing platform limitations hinder analysis of long DNA fragments common in methylated areas.

Purpose of the Study:

• To develop a novel method, BRIF-seq, to overcome read distribution bias in single-cell DNA methylation analysis.
• To improve genome coverage and maximize the utility of long DNA segments for methylome studies.

Main Methods:

• Bisulfite-converted randomly integrated fragments sequencing (BRIF-seq) was developed and applied to single microspores of maize.
• High-throughput sequencing was performed to assess DNA methylation states (CG, CHG, CHH) across the maize genome.

Main Results:

• BRIF-seq demonstrated higher rates of read mapping and improved genome coverage compared to scBS-seq.
• Reads generated by BRIF-seq were more evenly distributed across the genome, including highly methylated regions.
• Maize microspore methylome analysis revealed similar methylation rates within tetrads but significant differences among tetrads, suggesting potential non-simultaneous epigenetic reprogramming.

Conclusions:

• BRIF-seq effectively addresses read distribution bias and enhances genome coverage for single-cell methylome analysis.
• The method is suitable for diverse species, including those with highly methylated and repetitive genomes like maize.
• Findings suggest potential for non-simultaneous methylation reprogramming events during microsporogenesis.