Comparison of SureSelect and Nextera Exome Capture Performance in Single-Cell Sequencing

Area of Science:

• Genomics and Molecular Biology
• Cancer Research
• Single-Cell Analysis

Background:

• Single-cell sequencing enables detailed examination of rare cells like circulating tumor cells.
• Single-cell whole exome sequencing (scWES) is crucial for understanding cancer at a single-cell level.
• Limited knowledge exists regarding the performance of different exome capture kits for scWES.

Purpose of the Study:

• To compare the performance of Agilent SureSelectXT (AGL) and Nextera rapid capture (NXT) exome capture kits for scWES.
• To evaluate data quality and variant calling accuracy between AGL and NXT in scWES applications.

Main Methods:

• DNA libraries were generated from 192 single cells isolated from melanoma spheres using Fluidigm C1.
• Twelve high-yield cells underwent dual-exome capture and sequencing using both AGL and NXT kits in parallel.
• Performance was assessed through mapping rates, coverage uniformity, capture efficiency, and variant calling accuracy.

Main Results:

• AGL demonstrated superior performance over NXT in coverage uniformity, mapping rates, exome capture rates, and reduced PCR duplicates.
• AGL showed better germline variant calling accuracy, with higher overlap with dbSNP and appropriate transition-transversion ratios.
• AGL-based scWES achieved high positive predictive values and good sensitivity for somatic mutation detection compared to bulk sequencing.

Conclusions:

• Agilent SureSelectXT (AGL) is a viable and high-performing option for single-cell whole exome sequencing (scWES).
• AGL yields superior data quality compared to the Fluidigm protocol utilizing NXT, especially when sufficient DNA is available.
• This finding enhances the toolkit for detailed genomic analysis of rare cells in cancer research.