Defining immune landscape and clonal expansion in pre-stage multiple myeloma patients by mitochondrial single nucleotide polymorphisms (SNPs) enrichment

Abstract

ABSTRACT Multiple myeloma (MM) is a bone marrow plasma cell malignancy that is preceded by an asymptomatic condition called Smoldering Multiple Myeloma (SMM). Patients with SMM are typically observed until progression to overt MM. Early treatment at the SMM stage can prolong patient progression-free survival, presumably due to strong immune responses that control disease. While single-cell RNA-sequencing enables the study of multiple cell populations in a single experiment, it cannot resolve lineages within populations, and thus identify concerted clonal expansions. Parallel sequencing of the T- or B-cell receptors can uncover clonal populations, however no such approach exists for NK cells, Monocytes and Dendritic cells. Parallel sequencing of the mitochondrial exome enables the identification of single nucleotide polymorphisms (SNPs) which function as inherent cell barcodes and allow for lineage tracing across all populations profiled in the experiment. Nevertheless, it is challenging to amplify mitochondrial transcripts from barcoded cDNA, due to the presence of limited material, the use of multiple primers, and amplification specificity issues. Here, we developed an experimental protocol to amplify barcoded mitochondrial cDNA obtained through the commercial kit for 5’-end single-cell RNA and VDJ sequencing by 10X Genomics. We show that a shorter forward primer that binds and extends the Partial Read 1 sequence, coupled with a 1:5 ratio of forward: reverse primer, and a two-step Polymerase Chain Reaction (PCR) approach, allow for the successful amplification of barcoded mitochondrial transcripts, which are compatible with standard Illumina next-generation sequencing platforms. This approach will allow us to identify clonal expansions across all immune cell populations and determine novel immune cell biomarkers of response to early therapy in patients with SMM, which we hope will aid towards preventing disease progression, organ damage, and ultimately death in this patient population.