Bisulphite sequencing in the presence of cytosine-conversion errors

Area of Science:

• Molecular Biology
• Epigenetics
• Genomics

Background:

• Tagmentation-based bisulfite sequencing offers high throughput and low input DNA requirements for epigenetic analysis.
• Previous observations noted an elevated rate of unmethylated cytosine non-conversion in certain tagmentation protocols.

Purpose of the Study:

• To investigate and characterize the phenomenon of unmethylated cytosine non-conversion during tagmentation-based bisulfite sequencing.
• To elucidate the underlying molecular mechanism responsible for these sequencing errors.

Main Methods:

• Detailed analysis of DNA fragmentation and adapter ligation steps in tagmentation protocols.
• Investigating transposase activity, including nicking and strand displacement, on DNA substrates.
• Development of a computational framework to correct for observed sequencing errors.

Main Results:

• The observed errors are consistent with single-strand nicks by the transposase, leading to strand displacement and incorporation of methylated cytosines.
• These errors do not impede biological conclusions when accounted for in downstream analysis.
• A Python package is provided to implement a framework for error correction.

Conclusions:

• Tagmentation bisulfite sequencing errors can be understood and corrected computationally.
• The scalability of tagmentation protocols must be weighed against the effort required for error correction in experimental design.