Implementing Hifi Sequencing for Enhanced Genomic and Methylome Characterization in Multiple Myeloma
Authors: Han Seungbin , Afrin Nazia , Tolhuis Bas , Truger Marietta , Raab Peter , Munawar Umair , Vogt Cornelia , Kurian Shilpa , Nerreter Silvia , Zhou Xiang , Haferlach Claudia , Einsele Hermann , Waldschmidt Johannes , Rasche Leo , Kortüm K. Martin 
Published: December 2024

Introduction:

Over recent years, the introduction and scalability of next-generation sequencing technologies have significantly expanded our knowledge of the genetic underpinnings of multiple myeloma (MM), a heterogeneous hematologic malignancy characterized by clonal plasma cell proliferation in the bone marrow. At present, cost-effective short-read technologies are extensively utilized for sequencing. Nevertheless, these platforms are limited in their ability to fully capture repeated regions and structural variants. Progress in long-read sequencing technologies, including HiFi-sequencing (HiFi), produces extended reads with increased precision, enabling more comprehensive and continuous genome assemblies. Another notable benefit of HiFi is its ability to integrate genome and methylome data, which seems increasingly needed given that epigenetic regulation has been identified as a major driver of resistance in MM.

Methods:

We collected CD138+ cells from the bone marrow of four newly diagnosed and nine relapsed / refractory MM patients. Sequencing of all samples was performed with a coverage of 30x using HiFi sequencing. In addition, we conducted short-read sequencing with 100x coverage to facilitate a direct comparison between the two approaches. We performed in-depth coverage analysis using mosdepth to compare the read mappability of the two sequencing technologies. Comparative downstream analysis, with a specific focus on structural variations (SVs), single nucleotide variations (SNVs), and methylome analysis was performed.

Results:

The goal of this study was to conduct a technical feasibility analysis to examine genome coverage as well as detection of structural variants (SVs) and single nucleotide variants (SNVs) by HiFi long-read sequencing and short-read sequencing in a cohort of MM patients with heterogeneous disease activity. As anticipated, a mean genome coverage of 30.1x was achieved by HiFi-sequencing across all samples. Read length was consistently &gt;10,000 bp with a mean of 13,047 bp (range: 10,503-14,836). The median quality value (QV) for each base in the sequence was reassuring, with values ranging between 34 and 40. An average of 5,658,975 regions with high coverage (each region = 500 bp) were seen by HiFi sequencing as compared to 5,620,710 regions by short-read sequencing , resulting in a difference of 19.1 Mbp with improved coverage. By applying ClairS-TO, a deep-learning algorithm to uncover somatic mutations, we could observe that the HiFi technology could detect more somatic variants (n = 151,555) as compared to the short-read approach (n = 129,921). Structural variant calling and analysis are currently ongoing and will be performed by established tools for long-read vs. short-read analysis. Furthermore, we acquired healthy plasma cells from 13 individuals and sequenced with HiFiseq. The metyhlome and genome of these samples will serve as the reference for further comparison analysis with multiple myeloma patient samples through which we will provide insights into the MM cells specific genome-wide methylattion profile as well as genomics events.

Summary:

In summary, we here report on the feasibility of HiFi sequencing for enhanced genomic and methylome characterization in MM. Our final analysis is ongoing and updated results will be presented at the meeting.