Linked-read whole-genome sequencing resolves common and private structural variants in multiple myeloma.

REGULAR ARTICLE Linked-read whole-genome sequencing resolves common and private structural variants in multiple myeloma ~a-Pe
rez,1,2 Nicolai Frengen,1,2 Julia Hauenstein,1,2 Charlotte Gran,2,3 Charlotte Gustafsson,1,2 Jesper Eisfeldt,4,5 Luc
ıa Pen
Olsen,8 Ann Wallblom,2,3 Aleksandra Krstic,9 Philip Ewels,8 Marcin Kierczak,6 Fanny Taborsak-Lines,7 Remi-Andre 4,10 1,2,11 Anna Lindstrand, and Robert Månsson 1 Department of Laboratory Medicine, 2Center for Hematology and Regenerative Medicine, 3Department of Medicine, and 4Department of Molecular Medicine and Surgery, Karolinska Institutet, Stockholm, Sweden; 5Science for Life Laboratory, Karolinska Institutet Science Park, Stockholm, Sweden; 6Department of Cell and Molecular Biology, National Bioinformatics Infrastructure Sweden, Science for Life Laboratory, Uppsala University, Uppsala, Sweden; 7Division of Gene Technology, Royal Institute of Technology, Stockholm, Sweden; 8Department of Biochemistry and Biophysics, Science for Life Laboratory, Stockholm University, Stockholm, Sweden; and 9Department of Clinical Pathology and Cytology, 10Department of Clinical Genetics, and 11Department of Hematology, Karolinska University Hospital, Stockholm, Sweden Key Points   Linked-read WGS can be performed without DNA purification and allows for resolution of the diverse structural variants found in MM. Linked-read WGS can, as a standalone assay, provide comprehensive genetics in myeloma and other diseases with complex genomes. Multiple myeloma (MM) is an incurable and aggressive plasma cell malignancy characterized by a complex karyotype with multiple structural variants (SVs) and copynumber variations (CNVs). Linked-read whole-genome sequencing (lrWGS) allows for refined detection and reconstruction of SVs by providing long-range genetic information from standard short-read sequencing. This makes lrWGS an attractive solution for capturing the full genomic complexity of MM. Here we show that high-quality lrWGS data can be generated from low numbers of cells subjected to fluorescence-activated cell sorting (FACS) without DNA purification. Using this protocol, we analyzed MM cells after FACS from 37 patients with MM using lrWGS. We found high concordance between lrWGS and fluorescence in situ hybridization (FISH) for the detection of recurrent translocations and CNVs. Outside of the regions investigated by FISH, we identified .150 additional SVs and CNVs across the cohort. Analysis of the lrWGS data allowed for resolution of the structure of diverse SVs affecting the MYC and t(11;14) loci, causing the duplication of genes and gene regulatory elements. In addition, we identified private SVs causing the dysregulation of genes recurrently involved in translocations with the IGH locus and show that these can alter the molecular classification of MM. Overall, we conclude that lrWGS allows for the detection of aberrations critical for MM prognostics and provides a feasible route for providing comprehensive genetics. Implementing lrWGS could provide more accurate clinical prognostics, facilitate genomic medicine initiatives, and greatly improve the stratification of patients included in clinical trials. Introduction Multiple myeloma (MM) is a hematological malignancy affecting terminally differentiated B lineage cells and is characterized by the accumulation of clonal plasma cells in the bone marrow.1 It has a complex genetic landscape thought to cause the clinical heterogeneity of the disease both in terms Submitted 29 November 2021; accepted 31 May 2022; prepublished online on Blood Advances First Edition 8 June 2022; final version published online 30 August 2022. DOI 10.1182/bloodadvances.2021006720. Omics data is deposited on a secure Swedish server and has been assigned a DOI (https://doi.org/10.17044/scilifelab.17049059.v1). Data access requests may be submitted to the corresponding author () or the Science for Life Laboratory Data Centre through the DOI link. 13 SEPTEMBER 2022 • VOLUME 6, NUMBER 17 The full-text version of this article contains a data supplement. © 2022 by The American Society of Hematology. Licensed under Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International (CC BY-NCND 4.0), permitting only noncommercial, nonderivative use with attribution. All other rights reserved. 5009 of treatment response and overall outcome.2 The introduction of novel treatments has significantly improved survival.3 However, despite these advances, the disease essentially remains incurable, and patients with high-risk aberrations continue to display a poor outcome.3-5 Therefore, identifying high-risk patients in the context of developing therapy regimens and understanding underlying disease biology to find novel venues for treatment remain critical to improve overall outcome. On the basis of primary genetic events, MM is largely divided into hyperdiploid (HRD) and non-HRD cases.2,6 In this division, HRD MM is characterized by multiple trisomies of odd-numbered chromosomes, whereas non-HRD MM is associated with immunoglobulin heavy chain (IGH) translocations. The most common IGH translocations include t(4;14), t(11;14), t(6;14), t(14;16), and t(14;20), which, via colocalization with strong Em and 39 regulatory region (RR) IGH enhancers,7 cause the dysregulation of MMSET/FGFR3, CCND1, CCND3, MAF, and MAFB, respectively. Traditionally, these primary aberrations, together with common secondary events linked to poor outcome (including deletion of 17p, amplification of 1q21, and MYC translocation), have been investigated in clinical routine using fluorescence in situ hybridization (FISH). Despite this seemingly simple dichotomy of initiating events, next-generation sequencing has revealed a complex landscape of genetic aberrations.5,8-16 This landscape often comprises an array of secondary genetic aberrations, with frequent copy-number variations (CNVs), single-nucleotide variants (SNVs), and structural variants (SVs) resulting from templated insertions, focal amplifications, chromoplexy, chromothripsis, or other complex rearrangements.12,13,15-17 Collectively, these aberrations affect plasma cell differentiation, cellcycle regulation, DNA repair, and multiple signaling pathways.2 Major efforts are being made to exploit targetable aberrations, which, together with high-throughput sequencing–based genomics, create the possibility of using personalized medicine strategies for the treatment of MM.2,18-20 Linked-read whole-genome sequencing (lrWGS) is a developing technology that allows for the creation of synthetic long reads from conventional short-read sequencing. Linked-read data are achieved by generating groups of reads or read clouds originating from a single high molecular weight (HMW) DNA molecule, which all carry a common barcode linking them together.21-24 Mapping the read clouds together and subsequently leveraging the existence of SNVs within the read clouds allow for improved mapping and haplotype reconstruction (...truncated)