Epigenetic sequencing using TAPS
Drs Chunxiao Song and Benjamin Schuster-Böckler have developed a new, more sensitive detection method for methylated DNA with potential for use as a liquid biopsy test for cancer.
For earlier detection of cancer, researchers are seeking to develop methodologies that are less invasive than current diagnostic tests, which would allow them to be performed on more people and at a higher frequency. Liquid biopsies, taking advantage of cancer-specific biomarkers such as tumour-derived DNA in the blood, urine or saliva, are an attractive opportunity for a minimally invasive diagnostic assay. The challenge for the field is to make these detection methods sensitive and specific enough. To be used as a reliable cancer test, assays need to be able to detect the very low levels of circulating tumour DNA (ctDNA) in the blood and differentiate this from DNA shed into the blood from normal cells.
One approach to this challenge is to sequence the circulating DNA to detect cancer-specific mutations. However, relying on single or a few mutations, or DNA copy number variations alone, may be challenging for cancers with strong inter-patient and intra-tumour heterogeneity. An alternative approach is to look for cancer-specific patterns of epigenetic modifications, such as methylation and hydroxymethylation of the cytosine base in DNA (5mC and 5hmC, respectively). These modifications have important roles in gene silencing and other cellular regulatory processes but are frequently altered in cancer cells and maintained in tumour DNA that is released into the blood. An advantage of methylation profiling is that the usable information is spread across hundreds of sites, potentially increasing the chances of detecting evaluable profiles at low ctDNA concentration. Epigenetic signals may also provide cancer type-specific information, as Dr Chunxiao Song has shown in lung, pancreatic and liver cancer1, important in an early detection screening programme.
To date however, the clinical application of epigenetic profiling with low-input circulating DNA has been limited by its dependency on bisulphite sequencing. Bisulphite is a harsh chemical that causes severe degradation of the DNA sample, thus lowering sensitivity. Further, bisulphite sequencing detects 5-methylcytosine (5mC) and 5-hydoxymethylcytosine (5hmC) indirectly, converting all non-modified cytosine to thymine. This decreases sequencing complexity, reducing sequencing and mapping quality, and increasing sequencing costs. To overcome these issues, Drs Chunxiao Song and Benjamin Schuster-Böckler have developed a bisulphite-free and base-resolution sequencing method, TET-assisted pyridine borane sequencing (TAPS)2, for detection of 5mC and 5hmC.
Figure: Overview of the TAPS chemical biology method for bisulphite-free, base-resolution, and direct sequencing of 5-methylcytosine (5mC) and 5-hydroxymethylcytosine (5hmC). Ten-eleven translocation (TET) enzyme oxidation of 5mC and 5hmC to 5-carboxylcytosine (5caC) is followed by pyridine borane reduction of 5caC to dihydrouracil (DHU). Subsequent polymerase chain reaction (PCR) converts DHU to thymine, enabling a C-to-T transition of 5mC and 5hmC. By comparing the treated and untreated sequences, it is possible to identify the cytosine bases that have been converted to thymine during TAPS and therefore those that were modified by 5mC and 5hmC.
TAPS biochemistry selectively and directly converts 5mC and 5hmC to thymine in a mild reaction that preserves DNA integrity and is effective at very low DNA concentrations. Due to its unique direct mechanism, TAPS generates fewer errors and is cheaper than bisulphite sequencing. Computationally, TAPS data are also >3 times faster to process than bisulphite sequencing data. Of note, TAPS can also detect genetic mutations and copy number variations.
TAPS is now being tested in a variety of cancers, including oesophageal, pancreatic and liver cancers with the support of the NIHR Oxford Bioemedical Research Centre, Ludwig Cancer Research (Oxford Branch), Chunxiao Song’s CRUK-OHSU award and Ellie Barnes' DeLIVER programme.
1. Song CX, Yin S, Ma L, et al. 5-Hydroxymethylcytosine signatures in cell-free DNA provide information about tumor types and stages. Cell Res 2017;27(10):1231-42. doi: 10.1038/cr.2017.106
2. Liu Y, Siejka-Zielinska P, Velikova G, et al. Bisulfite-free direct detection of 5-methylcytosine and 5-hydroxymethylcytosine at base resolution. Nat Biotechnol 2019 doi: 10.1038/s41587-019-0041-2