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Defining clonal plasticity using whole exome sequencing in single cells in an index case of amp1q21 multiple myeloma

Defining clonal plasticity using whole exome sequencing in single cells in an index case of amp1q21 multiple myeloma
Defining clonal plasticity using whole exome sequencing in single cells in an index case of amp1q21 multiple myeloma
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.
2152-2650
Sahota, Surinder
66c1457f-cba2-4c49-9c8c-fcee0748b6b8
Bryant, Dean
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Weston-Bell, Nicola
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Tapper, William
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Bolomsky, Arnold
c05601d7-6b96-4d70-acc8-eb035201e3be
Zojer, Niklas
88a51a4d-0b56-4832-bc0f-f102fc49d656
Collins, Andrew
7daa83eb-0b21-43b2-af1a-e38fb36e2a64
Sahota, Surinder
66c1457f-cba2-4c49-9c8c-fcee0748b6b8
Bryant, Dean
10ed83e8-8080-4d9c-bba5-df9d4eec3a10
Weston-Bell, Nicola
c99c8c28-519f-4c3e-bd8c-679995a0472e
Tapper, William
9d5ddc92-a8dd-4c78-ac67-c5867b62724c
Bolomsky, Arnold
c05601d7-6b96-4d70-acc8-eb035201e3be
Zojer, Niklas
88a51a4d-0b56-4832-bc0f-f102fc49d656
Collins, Andrew
7daa83eb-0b21-43b2-af1a-e38fb36e2a64

Sahota, Surinder, Bryant, Dean, Weston-Bell, Nicola, Tapper, William, Bolomsky, Arnold, Zojer, Niklas and Collins, Andrew (2017) Defining clonal plasticity using whole exome sequencing in single cells in an index case of amp1q21 multiple myeloma. Clinical Lymphoma, Myeloma & Leukemia, 17 (1), [e108]. (doi:10.1016/j.clml.2017.03.196).

Record type: Meeting abstract

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.

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More information

Accepted/In Press date: 31 December 2016
e-pub ahead of print date: 22 April 2017
Published date: 2017
Venue - Dates: 16th International Myeloma Workshop, , New Delhi, India, 2017-03-01 - 2017-03-04

Identifiers

Local EPrints ID: 417881
URI: http://eprints.soton.ac.uk/id/eprint/417881
ISSN: 2152-2650
PURE UUID: f780af9b-e098-4fd1-9645-e951f51a8f19
ORCID for Nicola Weston-Bell: ORCID iD orcid.org/0000-0003-0075-7276
ORCID for William Tapper: ORCID iD orcid.org/0000-0002-5896-1889
ORCID for Andrew Collins: ORCID iD orcid.org/0000-0001-7108-0771

Catalogue record

Date deposited: 16 Feb 2018 17:30
Last modified: 10 Nov 2021 02:57

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Contributors

Author: Surinder Sahota
Author: Dean Bryant
Author: Nicola Weston-Bell ORCID iD
Author: William Tapper ORCID iD
Author: Arnold Bolomsky
Author: Niklas Zojer
Author: Andrew Collins ORCID iD

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