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Comparative analysis of single-cell parallel sequencing approaches in oocyte application.
Yan Qian1, Jinyue Liao1, Ashley Hoi Ching Suen1
1Developmental and Regenerative Biology Program, School of Biomedical Sciences, The Chinese University of Hong Kong, Shatin, N.T., Hong Kong Special Administrative Region, China.
The International Journal of Biochemistry & Cell Biology
|December 12, 2018
Summary
Comparing single-cell parallel sequencing methods reveals trade-offs: the beads-based approach maximizes mRNA capture but loses DNA, while the non-beads-based approach preserves DNA at the expense of some mRNA. Single-cell whole genome bisulfite sequencing is recommended for methylome mapping.
Area of Science:
- Genomics and Epigenetics
- Single-cell analysis technologies
Background:
- Single-cell parallel sequencing enables simultaneous analysis of genetic/epigenetic variations and gene expression within individual cells.
- Two primary methods exist for separating DNA and RNA from single cells: beads-based and non-beads-based approaches.
- Systematic performance differences between these single-cell parallel sequencing methods are not well-established.
Purpose of the Study:
- To systematically compare the performance of beads-based and non-beads-based methods for single-cell parallel sequencing.
- To evaluate the impact of each method on transcriptome and methylome profiling from the same single cell.
- To provide guidance for selecting appropriate single-cell parallel sequencing strategies.
Main Methods:
- Simultaneous transcriptome and methylome profiling from single mouse oocytes.
- Comparative analysis of DNA and RNA yield and quality between beads-based and non-beads-based single-cell separation techniques.
- Evaluation of single-cell whole genome bisulfite sequencing (scWGBS) versus single-cell reduced representation bisulfite sequencing (scRRBS) for methylome mapping.
Main Results:
- The beads-based approach captured the maximum quantity of mRNA but resulted in inevitable DNA loss.
- The non-beads-based approach yielded more DNA due to intact nuclei but led to partial mRNA loss.
- Single-cell whole genome bisulfite sequencing (scWGBS) is recommended over scRRBS for generating single-cell methylome maps due to higher sequencing coverage.
Conclusions:
- The choice between beads-based and non-beads-based single-cell parallel sequencing involves a trade-off between DNA and mRNA preservation.
- Single-cell whole genome bisulfite sequencing is a preferred method for comprehensive single-cell methylome analysis.
- This study provides foundational insights for optimizing single-cell parallel sequencing protocols and future technological development.
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