Ultra-deep whole-genome sequencing of cell-free DNA recovers most bone marrow-derived mutations in newly diagnosed multiple myeloma

Abstract Introduction Cell-free DNA whole-genome sequencing (cfWGS) offers a non-invasive alternative to molecular profiling of multiple myeloma (MM), potentially reducing reliance on invasive bone marrow (BM) biopsies. Standard-depth (~30–40×) cfWGS reliably estimates tumor fraction and detects broad copy number alterations, but often fails to capture somatic single-nucleotide variants (SNVs) present at low variant allele frequencies (VAFs) due to tumor subclonality or low tumor shedding. Recent studies in solid tumors suggest that pushing cfWGS to ultra-deep coverage (≥100×) can rescue low-frequency somatic mutations, yet this has not been explored in MM. With sequencing costs declining, ultra-deep cfWGS has potential as a comprehensive tool for MM genomic profiling directly from peripheral blood (PB) cfDNA. To determine whether ultra-deep cfWGS can match the mutational yield of BM WGS and capture actionable variants, we compared standard-depth (40×) Illumina cfWGS with 150× cfWGS (targeted 200×) generated using the Ultima Genomics platform. Methods Paired BM and PB cfDNA samples with matched buffy coat germline controls were collected at diagnosis from 11 transplant-eligible patients enrolled in the Multiple Myeloma Molecular Monitoring (M4) study (n=8) and IMMAGINE study (n=3). DNA from BM CD138+ selected cells underwent 30-80× WGS on Illumina NovaSeq 6000 with somatic variants called versus a matched normal using MuTect2. Matched cfDNA libraries were prepared from a median of 20ng (range 20-83ng) of cfDNA extracted from 10-20mL of plasma for Illumina, and from 50ng (range 22-150ng) for Ultima. Libraries were sequenced to 40× on Illumina (aligned with BWA-MEM/GATK; variants force-called using MRDetect) and 150× (targeted 200×) on Ultima Solaris (prepared with PPM-Seq; variants force-called via Ultima's ‘Single Read SNV’ pipeline). Tumor fractions were estimated using ichorCNA. Results At diagnosis (median age 57, range 41–75 years; 6 male, 5 female), 4 patients were high risk, 2 standard, and 5 unknown. Subtypes were 4 IgG, 5 IgA, 1 light-chain only, and 1 unknown; ISS stages: I (n=3), II (n=6), III (n=1), and unknown (n=1). BM WGS identified a median of 3,087 somatic SNVs (range 467–4,059). Matched cfDNA tumor fraction was 7.6% (range 4.5-33.7%). Median deduplicated genome-wide coverage was 49× (range 25–57×) for Illumina and 155× (range 116–320×) for Ultima cfWGS, with median alignment rates of 98.4% and 99.0%, respectively. Duplication rates were higher for Ultima (median 20.2%, range 13.3–21.3%) than Illumina (median 8.5%, range 5.5–17.4%), consistent with increased sequencing depth. While this showed diminishing returns on efficiency at higher depths, coverage was sufficient to recover low-VAF mutations. Ultra-deep 150× cfWGS recovered significantly more somatic SNVs compared to 40× cfWGS (median 2,571 vs. 1,861; median increase of 21%, range -2–87%; paired t-test, p < 0.01). Compared to BM tumor DNA, 150× cfWGS recovered a median of 88% of somatic mutations (range, 67–97%) versus 66% (range, 36–91%) at 40×. Notably, 150× cfWGS detected 69% (11/16) of OncoKB-annotated oncogenic or likely oncogenic variants (including NRAS p.Q61R, KRAS p.A146V, and TP53 p.M237I), vs. 31% at 40×. Mutation recovery gains with ultra-deep sequencing were inversely correlated with cfDNA tumor fraction (Spearman’s ρ = –0.83; p < 0.01), with the greatest improvement (median 73%, range 33–87%) in cases with low (≤6%) cfDNA tumor fraction. Among variants present at ≥7.5% VAF in Ultima data, 4 of 5 actionable mutations were also detected at 40×, indicating strong concordance for high-burden alterations. Seven additional actionable mutations were uniquely identified at a 150× coverage, predominantly at low allele frequencies (≤6%, median VAF 3.8%, range 1.7–13.2%). Conclusions Ultra-deep cfWGS at 150× using Ultima sequencing recovered over twice as many actionable somatic mutations compared to 40× Illumina, despite lower sequencing costs in this pilot cohort. This tumor-informed analysis demonstrated that somatic mutations identified by Illumina BM WGS could be reliably recovered from cfDNA sequenced on the Ultima platform. These findings support the feasibility of ultra-deep cfWGS for comprehensive genomic profiling in MM and motivate further evaluation of cfDNA-based analyses as less invasive alternatives to BM biopsies. Future work will validate these findings in larger cohorts and extend analyses to other variant types, including translocations.

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