Permutation-based significance analysis reduces the type 1 error rate in bisulphite sequencing data analysis of human umbilical cord blood samples

Autor: Essi Laajala, Viivi Halla-aho, Toni Grönroos, Ubaid Ullah Kalim, Mari Vähä-Mäkilä, Mirja Nurmio, Henna Kallionpää, Niina Lietzén, Juha Mykkänen, Omid Rasool, Jorma Toppari, Matej Orešič, Mikael Knip, Riikka Lund, Riitta Lahesmaa, Harri Lähdesmäki
Jazyk: angličtina
Rok vydání: 2022
Předmět:
Zdroj: Epigenetics, Vol 17, Iss 12, Pp 1608-1627 (2022)
Druh dokumentu: article
ISSN: 1559-2294
1559-2308
15592294
DOI: 10.1080/15592294.2022.2044127
Popis: DNA methylation patterns are largely established in-utero and might mediate the impacts of in-utero conditions on later health outcomes. Associations between perinatal DNA methylation marks and pregnancy-related variables, such as maternal age and gestational weight gain, have been earlier studied with methylation microarrays, which typically cover less than 2% of human CpG sites. To detect such associations outside these regions, we chose the bisulphite sequencing approach. We collected and curated clinical data on 200 newborn infants; whose umbilical cord blood samples were analysed with the reduced representation bisulphite sequencing (RRBS) method. A generalized linear mixed-effects model was fit for each high coverage CpG site, followed by spatial and multiple testing adjustment of P values to identify differentially methylated cytosines (DMCs) and regions (DMRs) associated with clinical variables, such as maternal age, mode of delivery, and birth weight. Type 1 error rate was then evaluated with a permutation analysis. We discovered a strong inflation of spatially adjusted P values through the permutation analysis, which we then applied for empirical type 1 error control. The inflation of P values was caused by a common method for spatial adjustment and DMR detection, implemented in tools comb-p and RADMeth. Based on empirically estimated significance thresholds, very little differential methylation was associated with any of the studied clinical variables, other than sex. With this analysis workflow, the sex-associated differentially methylated regions were highly reproducible across studies, technologies, and statistical models.
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