Nanopore sequencing reveals methylation changes associated with obesity in circulating cell-free DNA from Gottingen Minipigs

Profiling of circulating cell-free DNA (cfDNA) by tissue-specific base modifications, such as 5-methylcytosines (5mC), may enable the monitoring of ongoing pathophysiological processes. Nanopore sequencing allows genome-wide 5mC detection in cfDNA without bisulphite conversion. The aims of this study were: i) to find differentially methylated regions (DMRs) of cfDNA associated with obesity in Gottingen minipigs using Nanopore sequencing, ii) to validate a subset of the DMRs using methylation-specific PCR (MSP-PCR), and iii) to compare the cfDNA DMRs with those from whole blood genomic DNA (gDNA). Serum cfDNA and gDNA were obtained from 10 lean and 7 obese Gottingen Minipigs both with experimentally induced myocardial infarction and sequenced using Oxford Nanopore MinION. A total of 1,236 cfDNA DMRs (FDR<0.01) were associated with obesity. In silico analysis showed enrichment of the adipocytokine signalling, glucagon signalling, and cellular glucose homoeostasis pathways. A strong cfDNA DMR was discovered in PPARGC1B, a gene linked to obesity and type 2 diabetes. The DMR was validated using MSP-PCR and correlated significantly with body weight (P < 0.05). No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA. In conclusion, nanopore sequencing detected differential methylation in minute quantities (0.1-1 ng/mu l) of cfDNA. Future work should focus on translation into human and comparing 5mC from somatic tissues to pinpoint the exact location of pathology.

Automated summary

Profiling cfDNA using nanopore sequencing and tissue-specific base modifications enables the monitoring of ongoing pathophysiological processes. This study identified differentially methylated regions (DMRs) associated with obesity in Gottingen minipigs using nanopore sequencing, validated a subset of DMRs using MSP-PCR, and compared cfDNA DMRs with gDNA DMRs. The results showed 1,236 cfDNA DMRs associated with obesity, with enrichment of pathways related to adipocytokine signaling and glucose homeostasis. No DMRs intersected between cfDNA and gDNA, suggesting that cfDNA originates from body-wide shedding of DNA.