Defining Clonal Plasticity Using Whole Exome Sequencing in Single Cells in an Index Case of amp1q21 Multiple Myeloma

Abstract

Introduction: Genomic-wide analysis of multiple myeloma (MM) reveals subclonal evolution as a major mechanism in disease evolution and progression, and interrogation of the entire functional genome at the single cell (SC) level is essential to fully map intra- clonal variation (ICV) and competition as the driver mechanism. To progress this, we have sought to establish whole exome sequencing (WES) in SCs in MM, initially in an index case of amp1q21 MM. Methods: CD138+ tumour cells and CD3+ T-cells were isolated from a presentation case of amp1q21 MM. Single MM and T cells were isolated for single cell WES. Whole genome amplification was performed using Qiagen REPLI-g Mini kit, and exome capture was performed using Agilent SureSelect. Libraries were 90 bp PE sequenced on an Illumina HiSeq2000 (BGI, China). Data was produced for bulk (1000 cells) MM and T cells, 20 MM SCs and 5 T cell SCs. Fastq were aligned to hg19 ref sequence using NovoAlignMPI (v3.02.03). Variant calling was performed using VarScan (v2.3.6) and variants were annotated using ANNOVAR. High confidence variants were called in the bulk tumour WES by pairwise comparison with bulk germline WES and by annotation against variant databases to exclude germline variants. Multiple quality control measures were employed to minimise false positives. Results and Discussion: SC WES generated raw data that were similar to bulk WES, with comparable mapping to target (69-76% SC vs. 70% bulk) and mean fold coverage (56.8-59.1x vs. 59.7x bulk). On average, 82% of the exome was covered sufficiently for somatic variant (SV) calling (! 5x), matching seminal published SC WES studies (70-80%) (Hou et al., Cell, 2012; Xu et al., Cell, 2012). We called 33 high confidence potentially deleterious SVs in the bulk tumour, 21 of which were also called in ! 1 SC exomes. SVs include mutations in genes involved in plasma cell differentiation, the MAPK pathway and known pathways in origins of B cellmalignancy. Random SVs were validated by Sanger sequencing with 100% concordance in the bulk (15/15) and SCs (55/55). ICV was apparent from the SC exome variant data. Total variant counts varied considerably among SCs and most variant positions had at least several cells where no evidence of the variant existed. Bulk WES lacks crucial information: We called 23 variants in ! 2 SCs that were absent in the bulk. Of these, 7/7 amplifiable variants were re-sequenced to obtain 100% concordance. These variants are of high interest as they reveal a marked occurrence of subclonal mu- tations in the MM tumour that are not identified by bulk WES. They indicate that the mutational status of the MM genome may be substantially underestimated by bulk WES. Conclusion: We establish the feasibility of SC WES as a method for defining true intraclonal genetic variation as a driver of cancer plasticity in MM.