Optimized Workflow for Whole Genome and Transcriptome Next-Generation Sequencing of Single Cells or Limited Nucleic...

Area of Science:

• Genomics and Molecular Biology
• Next-Generation Sequencing Technologies
• Biotechnology

Background:

• Whole genome and transcriptome sequencing demand substantial nucleic acid quantities, often unavailable from single cells or scarce samples.
• Existing amplification methods like PCR have limitations, including high error rates and reduced library complexity.
• Degradation and contamination are significant challenges when working with minute amounts of nucleic acids.

Purpose of the Study:

• To present a high-fidelity multiple displacement amplification (MDA) workflow for accurate amplification of limited nucleic acid samples.
• To enable the preparation of high-quality next-generation sequencing (NGS) libraries from minute DNA or RNA inputs.
• To provide a comprehensive protocol for generating NGS libraries from single cells or small nucleic acid quantities for Illumina sequencing.

Main Methods:

• Utilized a high-fidelity multiple displacement amplification (HiFi MDA) technique for nucleic acid amplification.
• Developed a complete workflow encompassing cell lysis, nucleic acid amplification, and library preparation.
• Incorporated protocols for DNA/RNA quantification, purification, fragmentation, adapter ligation, and library normalization.

Main Results:

• Achieved accurate amplification of limited nucleic acid material using HiFi MDA.
• Enabled library preparation with high input starting material, reducing reliance on extensive PCR cycling.
• Demonstrated successful generation of high-quality NGS libraries from single cells and small DNA/RNA quantities.

Conclusions:

• HiFi MDA is a robust method for amplifying minute nucleic acid amounts, crucial for single-cell and low-input genomics.
• The described workflow provides a complete solution for preparing NGS libraries, overcoming limitations of traditional PCR-based amplification.
• This approach facilitates studies involving rare cell types, heterogeneous samples, and unculturable organisms, expanding the scope of genomic and transcriptomic analyses.