Report analysis of DNA
Methylation
High-throughput methods mostly rely on reference genomes, but many species lack such resources. Recently, Austrian researchers have developed a groundbreaking method to analyze DNA methylation without needing a reference genome. Their findings were published this week in Cell Reports.
The first author of the study, Johanna Klughammer from the CeMM Center for Molecular Medicine at the Austrian Academy of Sciences, and her team introduced RefFreeDMA—a software pipeline that simplifies reduced bisulfite sequencing (RRBS) data processing. This approach enables the inference of a genome from RRBS reads and identifies regions of differential methylation within a sample.
Their method leverages the non-random distribution of DNA methylation in vertebrates, where most methylation occurs at CpG sites. Using restriction enzymes like MspI and TaqI, which are insensitive to methylation, the researchers generated RRBS libraries by digesting DNA. These sequences were then used to build a comprehensive dataset.
The team tested their approach across nine species, including human, mouse, rat, cow, dog, chicken, salmon, and zebrafish. They optimized genome coverage and sample throughput using a 96-well plate system, increasing the number of covered CpG sites from 2.5 million to 4 million.
RefFreeDMA is a Linux-based pipeline that takes advantage of the unique features of RRBS reads, such as clear start and end points. By pooling all RRBS reads from a given species, the software infers a consistent sequence for each cluster. It also retains cytosine when both cytosine and thymine are present, potentially indicating methylated sites.
Once the inferred genome was created, the researchers compared it with a custom methylation detection script using BSMAP/RRBSMPA. They identified differentially methylated regions and exported top-ranked reads as FASTA/FASTQ files for further biological interpretation.
To validate their method, Klughammer and colleagues applied it to 44 samples from three species: human, bovine, and salmon. They found that the average methylation levels at CpG sites were similar between the referenced and reference-free approaches. Moreover, the inferred genome closely matched the reference genome, with 1,254,324 out of 1,522,786 deduced fragments aligning with the human genome.
"When we mapped the alignment positions of representative chromosomes, we found that the two methods showed strong agreement," the researchers noted. "For all samples and species, the DNA methylation values obtained by these two methods were highly consistent."
The researchers highlighted that this method can be widely applied not only to wild populations but also to agricultural and aquaculture samples, opening new possibilities for genomic research in diverse species.
Elisa
Kit
, elisakit,
Shanghai
Elisa
Experimental survey
, SOD
Kit
, IgG
Kit
, IgM
Kit
, WB
Experimental generation
,
Immunohistochemistry experiment
,
Free test
, GIBCO, AMRESCO-
Shanghai Xinfan Biological
10-Port Type-C Cabinet Charging Station
10-Port Quick Charger,10-Port Type-C Cabinet Charging Station,Type-C Cabinet Smart Charging Station
shenzhen ns-idae technology co.,ltd , https://www.best-charger.com