Method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter

• Main Authors: Stanislav A. Vasilyev, Anton V. Markov, Oksana Yu. Vasilyeva, Ekaterina N. Tolmacheva, Lada A. Zatula, Diana V. Sharysh, Daria I. Zhigalina, Victoria V. Demeneva, Igor N. Lebedev
• Format: Article
• Language: English
• Published: Elsevier 2021-01-01
• Series: MethodsX
• Subjects: NGS-based LINE-1 retrotransposon methylation analysis
• Online Access: http://www.sciencedirect.com/science/article/pii/S2215016121002387

The methylation index of the LINE-1 promoter is one of the most commonly used markers for assessing the global level of genome methylation in various human cells and tissues. We developed an NGS-based protocol for DNA methylation analysis of the LINE-1 retrotransposon promoter. This approach allows assessment of the DNA methylation index of 19 CpG sites in the LINE-1 promoter that have the highest tissue- or tumor-specific variability. The method provides a DNA methylation profile for analyzing either the methylation index of each CpG site independently or the mean DNA methylation index across the LINE-1 promoter. The results obtained using the developed method corresponded well to the level of methylation assessed using a commercially available kit for DNA pyrosequencing. In addition, our method provides much more information: 1) the DNA methylation profile of a significant part of the LINE-1 promoter and 2) the level of DNA methylation at individual LINE-1 loci in the genome. The method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter can be used in large-scale studies of the global level of genome methylation in normal human cells or tumors.To accomplish this, we modified the targeted massive parallel sequencing method based on 16S Metagenomic Sequencing Library Preparation protocol (Illumina, USA) by: • Introduction of the stage of bisulfite conversion of DNA. • Development of specific primers for the LINE-1 sequence.