Mapping the zebrafish brain methylome using reduced representation bisulfite sequencing

Reduced representation bisulfite sequencing (RRBS) has been used to profile DNA methylation patterns in mammalian genomes such as human, mouse and rat. The methylome of the zebrafish, an important animal model, has not yet been characterized at base-pair resolution using RRBS. Therefore, we evaluated the technique of RRBS in this model organism by generating four single-nucleotide resolution DNA methylomes of adult zebrafish brain. We performed several simulations to show the distribution of fragments and enrichment of CpGs in different in silico reduced representation genomes of zebrafish. Four RRBS brain libraries generated 98 million sequenced reads and had higher frequencies of multiple mapping than equivalent human RRBS libraries. The zebrafish methylome indicates there is higher global DNA methylation in the zebrafish genome compared with its equivalent human methylome. This observation was confirmed by RRBS of zebrafish liver. High coverage CpG dinucleotides are enriched in CpG island shores more than in the CpG island core. We found that 45% of the mapped CpGs reside in gene bodies, and 7% in gene promoters. This analysis provides a roadmap for generating reproducible base-pair level methylomes for zebrafish using RRBS and our results provide the first evidence that RRBS is a suitable technique for global methylation analysis in zebrafish.     

DNA methylome profiling identifies novel methylated genes in African American patients with colorectal neoplasia

The identification of genes that are differentially methylated in colorectal cancer (CRC) has potential value for both diagnostic and therapeutic interventions specifically in high-risk populations such as African Americans (AAs). However, DNA methylation patterns in CRC, especially in AAs, have not been systematically explored and remain poorly understood. Here, we performed DNA methylome profiling to identify the methylation status of CpG islands within candidate genes involved in critical pathways important in the initiation and development of CRC. We used reduced representation bisulfite sequencing (RRBS) in colorectal cancer and adenoma tissues that were compared with DNA methylome from a healthy AA subject’s colon tissue and peripheral blood DNA. The identified methylation markers were validated in fresh frozen CRC tissues and corresponding normal tissues from AA patients diagnosed with CRC at Howard University Hospital. We identified and validated the methylation status of 355 CpG sites located within 16 gene promoter regions associated with CpG islands. Fifty CpG sites located within CpG islands—in genes ATXN7L1 (2), BMP3 (7), EID3 (15), GAS7 (1), GPR75 (24), and TNFAIP2 (1)—were significantly hypermethylated in tumor vs. normal tissues (P < 0.05). The methylation status of BMP3, EID3, GAS7, and GPR75 was confirmed in an independent, validation cohort. Ingenuity pathway analysis mapped three of these markers (GAS7, BMP3 and GPR) in the insulin and TGF-β1 network—the two key pathways in CRC. In addition to hypermethylated genes, our analysis also revealed that LINE-1 repeat elements were progressively hypomethylated in the normal-adenoma-cancer sequence. We conclude that DNA methylome profiling based on RRBS is an effective method for screening aberrantly methylated genes in CRC. While previous studies focused on the limited identification of hypermethylated genes, ours is the first study to systematically and comprehensively identify novel hypermethylated genes, as well as hypomethylated LINE-1 sequences, which may serve as potential biomarkers for CRC in African Americans. Our discovered biomarkers were intimately linked to the insulin/TGF-B1 pathway, further strengthening the association of diabetic disorders with colon oncogenic transformation.     

Measuring the methylome in clinical samples: Improved processing of the Infinium Human Methylation450 BeadChip Array

The Infinium Human Methylation450 BeadChip Array^TM (Infinium 450K) is an important tool for studying epigenetic patterns associated with disease. This array offers a high-throughput, low cost alternative to more comprehensive sequencing-based methodologies. Here we compare data generated by interrogation of the same seven clinical samples by Infinium 450K and reduced representation bisulfite sequencing (RRBS). This is the largest data set comparing Infinium 450K array to the comprehensive RRBS methodology reported so far. We show good agreement between the two methodologies. A read depth of four or more reads in the RRBS data was sufficient to achieve good agreement with Infinium 450K. However, we observe that intermediate methylation values (20–80%) are more variable between technologies than values at the extremes of the bimodal methylation distribution. We describe careful processing of Infinium 450K data to correct for known limitations and batch effects. Using methodologies proposed by others and newly implemented and combined in this report, agreement of Infinium 450K data with independent techniques can be vastly improved.     

Region-based epigenetic clock design improves RRBS-based age prediction

Recent studies suggest that epigenetic rejuvenation can be achieved using drugs that mimic calorie restriction and techniques such as reprogramming-induced rejuvenation. To effectively test rejuvenation in vivo, mouse models are the safest alternative. However, we have found that the recent epigenetic clocks developed for mouse reduced-representation bisulphite sequencing (RRBS) data have significantly poor performance when applied to external datasets. We show that the sites captured and the coverage of key CpGs required for age prediction vary greatly between datasets, which likely contributes to the lack of transferability in RRBS clocks. To mitigate these coverage issues in RRBS-based age prediction, we present two novel design strategies that use average methylation over large regions rather than individual CpGs, whereby regions are defined by sliding windows (e.g. 5 kb), or density-based clustering of CpGs. We observe improved correlation and error in our regional blood clocks (RegBCs) compared to published individual-CpG-based techniques when applied to external datasets. The RegBCs are also more robust when applied to low coverage data and detect a negative age acceleration in mice undergoing calorie restriction. Our RegBCs offer a proof of principle that age prediction of RRBS datasets can be improved by accounting for multiple CpGs over a region, which negates the lack of read depth currently hindering individual-CpG-based approaches.     

Identification of potential ‘lifestyle-responsive’ epigenomic biomarkers in healthy women aged 18–40

Context: Human health is complex and multifaceted; there is a need for biomarkers that reflect the multidimensional nature of health.

Objective: To identify potential epigenomic biomarkers of health in women aged 18–40 participating in a six-month lifestyle intervention, next level health.

Materials and methods: Methylation data were obtained by reduced representation bisulphite sequencing of 21 female intervention participants as well as three non-participants. The Differential Methylation Analysis Package (DMAP) was used to investigate inter- and intra-individual variability and to identify potential targets of transient epigenetic control in the population studied.

Results: Eleven genes were identified as significantly differentially methylated post- intervention in all 21 participants. 1884 genomic locations were found to be differentially methylated amongst the total female population studied representing potential epigenomic biomarkers.

Conclusions: The ability to demonstrate epigenetic changes arising from a lifestyle intervention can provide key information on the relationship between gene regulation, human behaviour and health.     

基于微量DNA样本的简化甲基化文库构建方法研究

RRBS(简化甲基化测序)是一种有效研究DNA甲基化状态的测序方案。文库构建是该方案中最关键的实验步骤之一,RRBS文库构建往往因为酶切产物少,文库构建步骤繁多等因素,导致DNA起始量需要1 ug以上。然而很多来源于人的样本,如冰冻组织穿刺、石蜡切片、显微微切割等,都往往只能获得100 ng以下的DNA,无法满足常规RRBS文库构建的起始DNA总量要求,从而大大限制RRBS技术的应用范围。本研究主要探讨并设计了基于微量DNA样本(100 ng)的RRBS文库构建策略,主要通过优化Msp I酶切条件、DNA片段大小筛选、缩减反应步骤及减少DNA转移次数等技术手段,大大提高了DNA回收率,进而建立了2种有效使用微量DNA样品的RRBS文库构建方案(即EA-Method和WB-Method方案)。EA-Method方案在处理100 ng左右的DNA时,有经济、快速、高效的特点,但文库质量略差于WB-Method方案;WB-Method方案可将DNA起始量降低至10 ng,并且文库质量高。为验证WB-Method方法的可靠性,研究人员选取了3种志愿者样本(全血和口拭子),采用WB-Method同时进行常量和微量RRBS文库构建,并进行Illumina Hiseq平台测序,数据通过生物信息分析得到微量RRBS检测的CpG,CHG,CHH中C碱基的甲基化比例与常量RRBS结果一致。同时,该方法可以大大降低DNA损耗及损伤,因而该方法可将RRBS技术应用到更广泛的样本类型中去,有效拓展RRBS的研究领域。     

Measuring the methylome in clinical samples: Improved processing of the Infinium Human Methylation450 BeadChip Array

The Infinium Human Methylation450 BeadChip Array^TM (Infinium 450K) is an important tool for studying epigenetic patterns associated with disease. This array offers a high-throughput, low cost alternative to more comprehensive sequencing-based methodologies. Here we compare data generated by interrogation of the same seven clinical samples by Infinium 450K and reduced representation bisulfite sequencing (RRBS). This is the largest data set comparing Infinium 450K array to the comprehensive RRBS methodology reported so far. We show good agreement between the two methodologies. A read depth of four or more reads in the RRBS data was sufficient to achieve good agreement with Infinium 450K. However, we observe that intermediate methylation values (20–80%) are more variable between technologies than values at the extremes of the bimodal methylation distribution. We describe careful processing of Infinium 450K data to correct for known limitations and batch effects. Using methodologies proposed by others and newly implemented and combined in this report, agreement of Infinium 450K data with independent techniques can be vastly improved.