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. 2021 Jul 6:8:101445.
doi: 10.1016/j.mex.2021.101445. eCollection 2021.

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

Stanislav A Vasilyev  1 , Anton V Markov  1 , Oksana Yu Vasilyeva  1 , Ekaterina N Tolmacheva  1 , Lada A Zatula  2 , Diana V Sharysh  1 , Daria I Zhigalina  1 , Victoria V Demeneva  1 , Igor N Lebedev  1
Affiliations

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

Stanislav A Vasilyev et al. MethodsX. .

Abstract

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.

Keywords: DNA methylation; LINE-1 retrotransposon; Targeted bisulfite massive parallel sequencing.

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Conflict of interest statement

The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

Figures

Image, graphical abstract
Graphical abstract
Fig 1
Fig. 1
Method of targeted bisulfite massive parallel sequencing of the human LINE-1 retrotransposon promoter. a. Schematic representation of the targeted bisulfite massive parallel sequencing of 19 CpG sites in the LINE-1 promoter. The red frame indicates the analyzed CpG sites in this study, and the blue frame indicates the most frequently analyzed CpG sites as determined using the Qiagen pyrosequencing LINE-1 kit (explanations are in the text). The schematic profile of the plot was obtained from the LINE-1 methylation index across the 5’-UTR region in analyzed samples. b. Gel image of the results of the amplified LINE-1 sequences for five samples after the first PCR with attached adapters and the same samples with attached indices after the second PCR. M represents Sky-High DNA marker (Biolabmix, Russia) with size in bp indicated.
Fig 2
Fig. 2
Relationship between the average LINE-1 methylation index (%) measured by the introduced method (NGS) and bisulfite DNA pyrosequencing.
Fig 3
Fig. 3
Relationship between methylation indices of individual CpG sites as measured by the introduced method and average LINE-1 methylation index (%) as assessed by bisulfite DNA pyrosequencing (at positions 318, 321, and 328) trophoblast and ACTA2+ cells.
See this image and copyright information in PMC

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