Sample demultiplexing, multiplet detection, experiment planning and novel cell type verification in single cell sequencing

Identifying and removing multiplets from downstream analysis is essential to improve the scalability and reliability of single cell RNA sequencing (scRNA-seq). High multiplet rates create artificial cell types in the dataset. Sample barcoding, including the cell hashing technology and the MULTI-seq technology, enables analytical identification of a fraction of multiplets in a scRNA-seq dataset.We propose a Gaussian-mixture-model-based multiplet identification method, GMM-Demux. GMM-Demux accurately identifies and removes the sample-barcoding-detectable multiplets and estimates the percentage of sample-barcoding-undetectable multiplets in the remaining dataset. GMM-Demux describes the droplet formation process with an augmented binomial probabilistic model, and uses the model to authenticate cell types discovered from a scRNA-seq dataset.We conducted two cell-hashing experiments, collected a public cell-hashing dataset, and generated a simulated cellhashing dataset. We compared the classification result of GMM-Demux against a state-of-the-art heuristic-based classifier. We show that GMM-Demux is more accurate, more stable, reduces the error rate by up to 69×, and is capable of reliably recognizing 9 multiplet-induced fake cell types and 8 real cell types in a PBMC scRNA-seq dataset.