A medium throughput approach for single cell copy number variation sequencing towards efficient application in clinics

Abstract

Introduction: Single-cell copy number variation sequencing (scCNV-seq) is essential for genomic analysis. However, existing methods rely on whole-genome preamplification or special devices, suffering from high cost and biased CNV calling. These challenges are significant in clinical scenarios where cells are precious and limited.

Objectives: We aimed to provide a convenient, efficient, and accurate scCNV-seq method with a data analysis workflow for clinical application with confident output while eliminated limitations above.

Methods: We developed traceable medium-throughput scCNV-seq (msCNVS). This method directly labels cells in a microplate using barcode-containing modified Tn5 transposomes, enabling early pooling to streamline processing and circumvent preamplification. MsCNVS simultaneously barcoded currently 48 but scable up to 96 or 384 cells, followed by dual indexes (i5, i7) for library construction. A new bioinformatic process, two-dimensional fitting, was also outlined for accurate ploidy and copy number determination.

Results: MsCNVS reliably distinguished unique CNV patterns among 5 cell lines comprising 292 cells. Individual msCNVS profiles of 70 K562 cells exhibited high correlation (R = 0.90-0.98) with bulk sequence. Triplicates of HeLa bulk cells demonstrated near-perfect correlation (R = 0.99). No evident cross-contamination of reads was detected when mouse and human cells were processed together. The CNV profiles of 47 single cells from primary amniotic fluid cell cultures were validated via karyotyping and microarray. MsCNVS obtained superior coverage uniformity versus MDA and MALBAC, approached the level achieved by eMDA and DOP-PCR, and showed less fluctuation than PTA. Notably, msCNVS detected CNV deletions in two abnormal blastocysts (totaling 33 cells), including one exhibiting CNV mosaicism. It also revealed CNVs in 24 circulating tumor cells, 35 cancerous pleural effusion cells, and 152 patient-derived xenograft nuclei. All these results clearly set apart confident absolute CNVs from ambiguous fuzzy zones.

Conclusion: msCNVS promises a robust and highly efficient CNV-seq approach for precious and rare cells typically encountered in reproductive and cancer clinics.

Keywords: Embryo mosaicism; Medium throughput scCNV-seq; Tn5 transposome; circulated tumor cells (CTC); prenatal diagnosis (PD).