Development of High Coverage Mammalian Single-Cell Methylation Sequencing Techniques
CitationCao, Yunlong. 2019. Development of High Coverage Mammalian Single-Cell Methylation Sequencing Techniques. Doctoral dissertation, Harvard University, Graduate School of Arts & Sciences.
AbstractSingle-cell methylation studies are essential where cell-to-cell methylation variation play a key role. It is also critical when the cell samples subject to analysis are precious, rare or in minute amounts. Current techniques available to perform mammalian single-cell methylation analysis rely on bisulfite conversion. The DNA damage and loss caused by bisulfite conversion hinders the methylome coverage, and brings great difficulties in data analyzes, rendering high accuracy differential methylation comparison extremely difficult to perform between individual single cells.
In this thesis, the development of a new high coverage methylation sequencing technique is presented, sc-TAPS, for mammalian CpG analysis. sc-TAPS significantly reduces DNA loss and DNA damage, while maintaining a high methyl-conversion rate. Unlike bisulfite conversion, only methylated cytosines will be converted to uracil during sc-TAPS, therefore the DNA complexity remain intact, resulting in a more uniform amplification, higher mapping rate, and less PCR bias.
A highly efficient DNA methylation amplification has been developed to compensate for DNA loss during sc-TAPS. Methylome Replication Loops with Methyl-Transferase (MERLOT) utilizes methyl-transferase DNMT1 for maintenance of the methylation pattern. MERLOT amplifies the DNA template 1.7-fold per cycle. Over 95% methylation maintenance efficiency could be achieved by MERLOT while having a low de novo methylated rate of 1.5%. MERLOT combined with sc-TAPS (MERLOT-TAPS), gives by far the highest coverage of all mammalian single-cell whole methylome sequencing techniques known thus far.
The low DNA loss and damage property of sc-TAPS makes it possible to combine with many other techniques previously unable by bisulfite conversion. scATAC-TAPS can simultaneously profile nucleosome occupancy and DNA methylation in single cells. Simultaneous measurement of three-dimensional genome structure and DNA methylation could be achieved by combining dip-C with TAPS for mammalian diploid cells. Finally, scTAPS followed by multiplex PCR enables deep methylation sequencing for over 50 specific gene targets simultaneously.
Together, sc-TAPS is proved to become a new way for single-cell mammalian methylation analysis with high coverage and high accuracy. Many techniques can be applied along with sc-TAPS to build a toolbox, that could overcome a variety of difficulties previously unsolvable by bisulfite conversion.
Citable link to this pagehttp://nrs.harvard.edu/urn-3:HUL.InstRepos:42013082
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