Trends of all opioid-, heroin-, and fentanyl-involved nonfatal overdoses in the United States, October 2020 - April 2025.

Copy number alterations (CNAs) in somatic cells and copy number variations (CNVs) in germline are important chromosomal or chromosomal segmental changes that influence human traits. In recent years, bulk and single-cell sequencing techniques for CNV (or CNA; hereafter referred to collectively as CNV for simplicity) detection have greatly advanced our understanding of CNV properties in development, disease, and aging. However, compared with other single-cell sequencing technologies—particularly single-cell RNA sequencing (scRNA-seq)—applications of single-cell CNV sequencing (scCNV-seq) remain limited due to their low efficiency[, , ]. As CNV often causes early pregnancy failure and miscarriage[], an early application of CNV analysis was prenatal diagnosis (PD) which relied on chromosome microarray analysis (CMA) of amniotic fluid or chorionic villus cells and chromosome karyotyping. Later, revised methods based on single-nucleotide variation (SNV), including non-invasive prenatal testing (NIPT) that sequences cell-free DNA from maternal plasma and preimplantation genetic testing (PGT) that sequences biopsies of trophoblast cells have been developed for assessing Mendelian diseases []. While SNV sequencing and analysis are now well established in precision medicine, CNV sequencing and analysis remain underdeveloped and less frequently applied. It is proposed that CNVs can greatly improve the detection of malignant progression in some solid tumors and leukemias with much lower costs. In particular, CNV may identify more biomarkers for early screening of cancer and monitor and guide cancer therapy[,]. Although several studies have explored CNVs in circulating tumor cells (CTCs) or minimal residual disease (MRD, a small number of cancer cells left in the body after treatment, and these cells have the potential to cause relapse), clinical applications remain limited due to unsatisfactory robustness and efficiency in CTC isolation and scCNV detection [, , ]. Initial scCNV-seq methods rely on whole-genome pre-amplification (preWGA)[] of individual cells, followed by library construction and DNA sequencing. Multiple methods were developed for processing individual cells’ genomes after preWGA, including degenerate oligonucleotide-primed PCR (DOP-PCR)[], multiple displacement amplification (MDA)[], multiple annealing and looping-based amplification cycles (MALBAC)[], linear amplification via transposon insertion (LIANTI)[], and primary template-directed amplification (PTA)[,]. Notably, PTA presents extremely low bias and high accuracy for single cell whole genome amplification, therefore it shows widely used both in SNV and CNV detection. Nevertheless, techniques including PTA, MIDAS, LIANTI encounter multiple challenges while downstream CNV library construction from preWGA, for example, low throughput, high labor intensity, long processing times, and high costs. Moreover, biases introduced during preWGA cannot be adequately mitigated through computational methods[]. These should limit the application in clinics.