METHOD FOR COMBINED GENOME METHYLATION AND VARIATION ANALYSES

METHOD FOR COMBINED GENOME METHYLATION AND VARIATION ANALYSES

Researchers at Stanford have developed improved methods for combined analyses of epigenetics and nucleotide variation in single cells.

Single cell genomic sequencing has allowed characterization of the diversity and evolution of cells within tissues. Recent advances in whole genome amplification, such as primary template-directed amplification (PTA), reproducibly captures the genomes of single cells in a more uniform and accurate manner, significantly improving nucleotide variation detection and analyses. Methods to combine epigenomic profiling with genomic variation analyses in single cells would enable researchers to study the connections between cell state and somatic mutations in normal homeostasis and disease.

Stage of Research

The inventors have developed improved methods of identifying modified cytosines in genomic DNA in a biological sample. The method, scMethyl-PTA, includes isolation and deamination of genomic DNA of interest from single cells. Deamination results in the conversion of non-modified cytosines to uracil and modified cytosines (e.g., 5-methylcytosine) to thymine. The sample is then treated with uracil DNA glycosylase to remove uracil nucleotides before PTA-based genomic amplification and sequencing in order to reveal modified cytosines and single nucleotide variations. Bioinformatic data integration methods infer methylation state of genome regulatory regions based on the enrichment of cytosine to thymine variants.

Applications

Single cell genomic and epigenomic profiling for concurrent variant and methylation detection in the same cell

Advantages

scMethyl-PTA recovers over 90% of the genome and enables accurate genome-wide calling of methylation status, copy number, and other structural variants in the same single cells
PTA results in significantly improved single nucleotide variation (SNV) detection and lower rates of false positives over whole genome amplification methods
Preferential deamination of 5-methylcytosine creates low-density cytosine deamination events, leaving the remaining genome intact and increasing genome recovery over traditional bisulfite methods

Stage of Development

Research – in vitro

Publications

Gonzalez-Pena V, et al. Accurate genomic variant detection in single cells with primary template-directed amplification. 2021. PNAS. DOI: 10.1073/pnas.2024176118