GMM-Demux: sample demultiplexing, multiplet detection, experiment planning, and novel cell-type verification in single cell sequencing

Abstract

Identifying and removing multiplets are essential to improving the scalability and the reliability of single cell RNA sequencing (scRNA-seq). Multiplets create artificial cell types in the dataset. We propose a Gaussian mixture model-based multiplet identification method, GMM-Demux. GMM-Demux accurately identifies and removes multiplets through sample barcoding, including cell hashing and MULTI-seq. GMM-Demux uses a droplet formation model to authenticate putative cell types discovered from a scRNA-seq dataset. We generate two in-house cell-hashing datasets and compared GMM-Demux against three state-of-the-art sample barcoding classifiers. We show that GMM-Demux is stable and highly accurate and recognizes 9 multiplet-induced fake cell types in a PBMC dataset.

Spermidine dampens inflammation by directly inhibiting Th17 cytokine production through a PRDX1 associated antioxidant pathway

“…We established LPS-induced acute lung injury and live bacterial infection models in Rag2/II2rg double gene knockout mice, which are deficient in T cells, B cells and NK cells. 24 The Th17 cytokines were completely absent in these mouse according to RT-PCR (data not shown), confirming that T cell function were knocked out. According to both ALI (Figure 5A-C) and PA14 infection (Figure 5D-F) models, spermidine did not protect against inflammation in Rag2/II2rg double gene knockout mice.…”

“…To minimize batch effect between samples, we multiplex all the samples with cell hashing method 23 for spermidine treatment (SPT sper and LN sper) and untreated groups (SPT stim and LN stim) (Figure 6A). The results after de-multiplexing 24 show that, compared with the spermidine untreated T cell control group (SPT stim), the expression of Txn1 and Prdx1 genes were significantly increased in the spermidine treatment group (Figure 6B-C). Prdx1 encodes peroxidase 1 (PRDX1), which is an antioxidant enzyme that protects the cell by reducing hydrogen peroxide retention.…”

Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design

“…Existing demultiplexing approaches differ in their experimental procedures, computational methodology for demultiplexing, and demultiplexing accuracy. In barcoding-based approaches (e.g., MULTI-seq [ 16 ] and cell hashing [ 17 ], and GMM-Demux for doublet identification [ 18 ]), cells are experimentally tagged with universal oligonucleotides or antibodies together with sample-specific labels, which can give highly accurate demultiplexing performance. However, these approaches make sample preparation more complex and increase costs due to reagent purchases as well as additional library preparation and sequencing.…”

Genetic demultiplexing of pooled single-cell RNA-sequencing samples in cancer facilitates effective experimental design

“…In barcoding-based approaches (e.g. MULTI-seq [16] and cell hashing [17], and GMM-Demux for doublet identification [18]), cells are experimentally tagged with universal oligonucleotides or antibodies together with sample-specific labels, which can give highly accurate demultiplexing performance, but these approaches make sample preparation more complex and increase costs due to reagent purchases as well as additional library preparation and sequencing. Alternatively, genetic variation-based approaches rely only on natural genetic variation between samples from different individuals (such as single nucleotide polymorphisms, SNPs), which does not require additional experimental procedures at the single-cell level.…”

“…In barcoding-based approaches (e.g. MULTI-seq [16] and cell hashing [17], and GMM-Demux for doublet identification [18]), cells are experimentally tagged with universal oligonucleotides or antibodies together with sample-specific labels, which can give highly accurate demultiplexing performance. However, these approaches make sample preparation more complex, and increase costs due to reagent purchases as well as additional library preparation and sequencing.…”