Methylation Matters: A Beginner’s Guide to Bisulfite Sequencing

  • Published: 26 June 2025
Let’s talk about epigenetics! Since this is quite a wide topic, we are going to focus on a gold standard method for studying DNA methylation - bisulfite sequencing. In this post, we’ll break down how bisulfite sequencing works, why it matters in fields like cancer research and developmental biology, and what makes it such a cornerstone in the study of the epigenome.

What is bisulfite sequencing?

Bisulfite sequencing is the gold standard method for studying DNA methylation. Methylation is a key epigenetic marker – a chemical tag added to DNA, usually at cytosine bases (the 'C' in the DNA code). It doesn't change the DNA sequence itself, but it affects how genes are expressed, playing crucial roles in development, disease, and response to the environment. Bisulfite treatment chemically converts unmethylated cytosines into uracils (which are read as thymine 'T' after PCR), while methylated cytosines remain unchanged as they are chemically resistant to this conversion under standard conditions. By sequencing the DNA after this treatment and comparing it to the reference genome, we can pinpoint exactly which cytosines were originally methylated in the sample.

Why is bisulfite sequencing important?

It's important for basic research as it is responsible for establishing cell identity during development, regulating tissue-specific gene expression, and methylation patterns involved in X-chromosome inactivation and genomic imprinting. But it is also relevant clinically as aberrant DNA methylation patterns are linked to many human diseases, especially cancer, where they can silence tumor suppressor genes or activate oncogenes. Understanding these patterns helps us understand disease mechanisms, develop diagnostic biomarkers, and potentially find new therapeutic targets. It's also vital in developmental biology, aging research, and understanding how environmental factors influence our genes.

How does bisulfite sequencing work?

The core workflow involves a few key steps. First, you isolate DNA from your sample. Then comes the crucial bisulfite conversion step – treating the DNA with sodium bisulfite under specific conditions to convert those unmethylated cytosines to uracils. After conversion, the DNA is quite fragile and fragmented. The next critical step is PCR amplification to generate enough material for sequencing. You need to amplify these converted DNA fragments accurately. Finally, the amplified library is sequenced, and bioinformatics tools are used to align the reads back to the reference genome and determine the methylation status at each cytosine position.

What are the challenges with bisulfite sequencing?

There are some challenges with this method that should be considered. Firstly, the bisulfite treatment itself is harsh and degrades a significant portion of the DNA, often leading to low yields and requiring higher input amounts. Secondly, the conversion process can be incomplete, leaving some unmethylated cytosines unconverted, which leads to false-positive methylation calls. Thirdly, and very relevant to the PCR step, the DNA template now contains uracils. Standard high-fidelity DNA polymerases – the enzymes used for accurate PCR – often stall or stop when they encounter uracil bases, as they typically recognize it as damage. This leads to biased amplification, meaning some fragments amplify much better than others, skewing the results and making it hard to accurately represent the original methylation profile, especially from low-input samples.

How to overcome the amplification bias due to uracils?

It used to be that researchers had to choose: use a non-proofreading polymerase that can read uracils but makes more errors or use a proofreading (high-fidelity) polymerase that's accurate but stalls at uracils, leading to bias and low yields. This is where advancements in enzyme technology become critical. We at Solis BioDyne recognized this challenge and developed a novel high-fidelity DNA polymerase specifically engineered to overcome this issue.

What is Solis BioDyne’s solution?

We recently launched our new HiFi polymerase – the Solis HiFi Hot Start DNA Polymerase. It has some unique modifications in the uracil binding pocket of the enzyme and unlike standard HiFi enzymes, it efficiently reads through uracil bases in the template strand without stalling, while still maintaining high accuracy due to its proofreading capability. This directly addresses the amplification bias challenge in bisulfite sequencing workflows. Researchers can now achieve high-fidelity amplification of bisulfite-converted DNA, leading to more accurate methylation profiling, better library complexity, and reliable results even from challenging low-input or degraded samples like those often used in clinical research. This directly tackles the amplification bias issue, enabling more uniform amplification of bisulfite-converted DNA, which translates to higher quality data, better library complexity, and improved sensitivity from low-input samples. Although, there are a few other HiFi enzymes on the market capable of tolerating uracil nucleotides, there is already positive feedback from clients testing Solis HiFi polymerase in bisulfite sequencing workflows and we hope this is a valuable tool for researchers in this field.