Abstract Title: Medium throughput sCNV-seq and msRRBS towards clinic application
Abstract: Single-cell CNV sequencing (scCNV-seq) is essential for genomic analysis, yet existing methods are limited by high cost, biased calling, and reliance on whole-genome preamplification or specialized equipment - major barriers to clinical application where cell numbers are scarce. To address these challenges, we developed msCNVS, a traceable, medium-throughput method that directly labels cells in a microplate using barcoded Tn5 transposomes. This design enables early pooling, eliminates preamplification, and requires no specialized instruments. msCNVS is adaptable and currently processes up to 48 cells (scalable to 96–384), supported by a two-dimensional fitting algorithm for accurate CNV calling. Overall efficiency is improved by more than tenfold. msCNVS reliably distinguished CNV patterns across five cell lines (292 cells), showing high concordance with bulk sequencing (K562: R = 0.90–0.98; HeLa triplicates: R = 0.99) and no detectable cross-contamination. It achieved superior coverage uniformity over MDA and MALBAC, approached the levels of eMDA and DOP-PCR, and exhibited less fluctuation than PTA. Validation was confirmed using karyotyping and microarray in primary amniotic fluid cells. The method successfully detected CNV deletions in abnormal blastocysts (including one case of mosaicism), as well as CNVs in circulating tumor cells, cancerous pleural effusion cells, and patient-derived xenograft (PDX) nuclei. Together, these results establish msCNVS as a robust and efficient CNV-seq solution for precious and rare cells encountered in reproductive and cancer clinics. Similarly we have also developed a highly efficient approach for single cell DNA methylome sequencing: medium-throughput single-cell Reduced Representation Bisulfite Sequencing: msRRBS.
