Biology subjects

Ponnaluri, V. K. C.

Publications and source records attributed to Ponnaluri, V. K. C..

3 recordsLinked to original sources

bioRxiv2020

The SEQC2 Epigenomics Quality Control (EpiQC) Study: Comprehensive Characterization of Epigenetic Methods, Reproducibility, and Quantification

Cytosine modifications in DNA such as 5-methylcytosine (5mC) underlie a broad range of developmental processes, maintain cellular lineage specification, and can define or stratify cancer and other diseases. However, the wide variety of approaches available to interrogate these modifications has created a need for harmonized materials, methods, and rigorous benchmarking to improve genome-wide methylome sequencing applications in clinical and basic research. Here, we present a multi-platform assessment and a global resource for epigenetics research from the FDAs Epigenomics Quality Control (EpiQC) Group. The study design leverages seven human cell lines that are designated as reference materials and publicly available from the National Institute of Standards and Technology (NIST) and Genome in a Bottle (GIAB) consortium. These samples were subject to a variety of genome-wide methylation interrogation approaches across six independent laboratories, with a primary focus was on 5-methylcytosine modifications. Each sample was processed in two or more technical replicates by three whole-genome bisulfite sequencing (WGBS) protocols (TruSeq DNA methylation, Accel-NGS MethylSeq, and SPLAT), oxidative bisulfite sequencing (TrueMethyl), one enzymatic deamination method (EMseq), targeted methylation sequencing (Illumina Methyl Capture EPIC), and single-molecule long-read nanopore sequencing from Oxford Nanopore Technologies. After rigorous quality assessment and comparison to Illumina EPIC methylation microarrays and testing on a range of algorithms (Bismark, BitmapperBS, BWAMeth, and GemBS), we found overall high concordance between assays (R=0.87-R0.93), differences in efficency of read mapping and CpG capture and coverage, and platform performance. The data provided herein can guide continued used of these reference materials in epigenomics assays, as well as provide best practices for epigenomics research and experimental design in future studies.

genomics↗

bioRxiv2019

EM-seq: Detection of DNA Methylation at Single Base Resolution from Picograms of DNA

Bisulfite sequencing is widely used to detect 5mC and 5hmC at single base resolution. However, bisulfite treatment damages DNA resulting in fragmentation, loss of DNA and biased sequencing data. To overcome this, we developed Enzymatic Methyl-seq (EM-seq), an enzymatic based approach that uses as little as 100 pg of DNA. EM-seq outperformed bisulfite converted libraries in all metrics examined including coverage, duplication, sensitivity and nucleotide composition. EM-seq libraries displayed even GC distribution, improved correlation across input amounts as well as increased representation of genomic features. These data indicate that EM-seq is more accurate and reliable than whole genome bisulfite sequencing (WGBS).

genomics↗

bioRxiv2017

Hypermethylation of human DNA: Fine-tuning transcription associated with development

Tissue-specific gene transcription can be affected by DNA methylation in ways that are difficult to discern from studies focused on genome-wide analyses of differentially methylated regions (DMRs). We studied 95 genes in detail using available epigenetic and transcription databases to detect and elucidate less obvious associations between development-linked hypermethylated DMRs in myoblasts (Mb) and cell-and tissue-specific expression. Many of these genes encode developmental transcription factors and display DNA hypermethylation also in skeletal muscle (SkM) and a few heterologous samples (e.g., aorta, mammary epithelial cells, or brain) among the 38 types of human cell cultures or tissues examined. Most of the DMRs overlapped transcription regulatory elements, including canonical, alternative, or cryptic promoters; enhancers; CTCF binding sites; and long-noncoding RNA (lncRNA) gene regions. Among the prominent relationships between DMRs and expression was promoter-region hypermethylation accompanying repression in Mb but not in many other repressed samples (26 genes). Another surprising relationship was down-modulated (but not silenced) expression in Mb associated with DNA hypermethylation at cryptic enhancers in Mb although such methylation was absent in both non-expressing samples and highly expressing samples (24 genes). The tissue-specificity of DNA hypermethylation can be explained for many of the genes by their roles in prenatal development or by the tissue-specific expression of neighboring genes. Besides elucidating developmental epigenetics, our study provides insights into the roles of abnormal DNA methylation in disease, e.g., cancer, Duchenne muscular dystrophy, and congenital heart malformations.

genomics↗