DNA methylation analysis
gCore offers DNA methylation analysis using either long-read or short-read sequencing
DNA methylation plays a crucial role in stem cell and tissue differentiation, serving as the primary mechanism for defining and maintaining cell type. In mammalian cells, this process predominantly occurs in CpG islands, often found in gene promoters. These methylation marks are vital for determining the differentiation status of a cell, initiating a series of events that lead to cellular specialization, thereby influencing gene expression and cell function. Unlike proteins, which have short lifespans of hours or days, DNA methylation marks can persist for extended periods, spanning months to years. Consequently, analyzing DNA methylation is a pivotal method for accurately identifying cell type and differentiation status, capturing the initial events that specify cell identity. This analysis tool is particularly useful in the fields of stem cell and regenerative medicine research. In brain research, DNA methylation was implicated in long-term effects such as aging, long-term memory and addiction. Thus, DNA methylation analysis can be useful in stem cell, regenerative medicine and brain research.
Genomics Core at SCRM now offers two methodologies for DNA methylation analysis:
Genome-wide DNA Methylation Analysis Using NEB Kit and Short-Read Sequencing
Genome-wide DNA Methylation Analysis via Long-Read Sequencing (PacBio Revio)
See details and pricing below.
NEB and Illumina support
BioModal 5hmc and 5mC sequencing
The advantage of BioModal DUET EVOC technology, followed by short-read sequencing, is that it outputs 6-letter DNA sequences A, G, C, T, M (5mC), and H (5hmc). In other words, Biomodal sequencing produces the standard AGCT sequence, which includes 5hmC and 5mC modifications.BioModal technology is based on enzymatic conversion, which results in high accuracy.
UCSD pricing:
Batch: 6 samples, 17x coverage in each of three modes (normal AGCT, 5mC, 5hmC)
Library prep: $933 per sample
Sequencing: $$467 per sample (6 samples are pooled in one NovaSeq X Plus 25B Flowcell 300)
TOTAL: $1400 per sample
5 hydroxy methylcytosine (5hmC)
Recently, 5 hydroxy mC (5hmC) comprising about 10% of methylated cytosines was shown to play a distinct functional role in the brain and gene regulation. The chemistry of the currently used NEB kit (NEBNext Enzymatic Methyl-seq Kit) doesn't distinguish between 5hmC and 5mC, resulting in either being classified as 5mC. Similarly, PacBio Revio classifies 5hmC as 5mC. However, Nanopore sequencing recognizes 5hmC and identifies both 5hmc and 5mC correctly and distinctly.
Enzymatic Short-Read Methylation with NEB-Illumina Combination
The advantage of short reads by Illumina is that their prices recently decreased following the acquisition of the Illumina NovaSeqX+ by the UCSD IGM Genomics Center. The advantage of enzymatic over bisulfite sequencing is that bisulfite damages DNA, and its timing needs to be calibrated at every experiment to avoid excessive conversion, while enzymatic is gentle on DNA and offers higher specificity with no requirement for calibration.
You submit 5-250ng of purified genomic DNA per sample. gCore prepares the libraries and sequences them at IGM. You get back the sequencing data in which unmethylated C is converted to T. By comparing the data to the reference genome, the positions of methylated cytosines are determined. Additionally, bioinformatic analysis is also offered by gCore.
UCSD Pricing
$135 per library prep, including the reagents and labor
$565 for Illumina sequencing
Total: $700 per sample, including library and sequencing. DNA extraction and bioinformatics can be added separately.
The samples are sequenced in batches of 3-4 samples per PE150 lane. The current turnaround time is 3-4 weeks.
Bioinformatics: $120 per sample. Discounts are available for batches exceeding 8 samples.
Genome-wide DNA methylation analysis with PacBio Revio long-read sequencing.
Priced at $1900 per sample, this service includes high molecular weight DNA extraction, library preparation, sequencing with approximately 15x coverage, and comprehensive bioinformatic analysis.
UCSD pricing:
PacBio support