DOGMA-seq and multimodal, single-cell analysis in acute myeloid leukemia

Abstract

Acute myeloid leukemia (AML) is a complex cancer, yet advances in recent years from integrated genomics methods have helped improve diagnosis, treatment, and means of patient stratification. A recent example of a powerful, multimodal method is DOGMA-seq, which can measure chromatin accessibility, gene expression, and cell-surface protein levels from the same individual cell simultaneously. Previous bimodal single-cell techniques, such as CITE-seq (Cellular indexing of transcriptomes and epitopes), have only permitted the transcriptome and cell-surface protein expression measurement. DOGMA-seq, however, builds on this foundation and has implications for examining epigenomic, transcriptomic, and proteomic interactions between various cell types. This technique has the potential to be particularly useful in the study of cancers such as AML. This is because the cellular mechanisms that drive AML are rather heterogeneous and require a more complete understanding of the interplay between the genetic mutations, disruptions in RNA transcription and translation, and surface protein expression that cause these cancers to develop and evolve. This technique will hopefully contribute to a more clear and complete understanding of the growth and progression of complex cancers.

Keywords: Acute myeloid leukemia; DOGMA-seq; Leukemic stem cell; Multi-omics; Single cell analysis.

Fig. 1

The flow chart of the DOGMA-seq in AML.

Fig. 2

Overall protocol of DOGMA-Seq. (a) Cell after sorting for viable cells were stained with a Cite-seq antibody panel of interest (Totalseq-A), washed, followed by fixation of cells with either Formaldehyde (0.1%) or no fixation (DIG method), permeabilized using LLL buffer or DIG buffer—user-defined. The cells were counted and adjusted for cell numbers according to the 10x protocol and transposed according to the 10x genomics multi-ome kit protocol. Once the cells are transposed, the cells are ready to be loaded on a 10x chromium controller according to the manufacturer’s protocol. (b) Generation of GEMs (Gel Bead-in-Emulsion) to capture single cells was performed in the 10x chromium controller where the transposed cells, along with master mix (provided in the 10x genomics multi-ome kit), were introduced in the droplet which contains beads which has capture sequences for both RNA (poly dT) and DNA (capture sequence). The CITE antibodies also have polyA sequences which the beads will capture through polydT sequences similar to RNA. (c) Once the GEM is generated, it is subjected to simultaneous RT-PCR followed by template switch, transcript extension, and DNA extension PCR, where barcodes were introduced to RNA, DNA, and ADT tags. This was further amplified according to 10x multi-ome kit instructions. ADT primers (0.2 uM–1 ul) were added during the pre-amplification step for ADT tag amplification. (d) Upon completion of the Pre-amplification step, the ATAC, cDNA, and ADT tags were amplified. This library was subjected to SPRI clean-up, and instead of deleting it in the 10x multi-ome kit suggested volume, the DNA fragments were eluted in 100ul of EB buffer. This was further divided into three aliquots to generate ATAC, gene expression (GEX), and ADT libraries according to the protocol described.