Anti cd138 magnetic microbeads

The bone marrow (BM) aspirate samples were purified by positive selection of anti-CD138 magnetic microbeads (MiltenyiBiotec, Germany) in 141 NDMM patients. The other four patients without monoclonal plasma cells in BM used a biopsy sample of EMD. Genomic DNA was extracted from CD138+ cells using the QIAamp Blood DNA Mini Kit and biopsy samples using the GeneRead™ FFPE Kit (QIAGEN, Germany). AmpliSeq designed the MM panel for the Illumina Gene Assay with the Illumina Design Studio platform (https://designstudio.illumina.com/). It included 92 MM-related genes (Supplementary File 1), including TP53 (16 (link)–18 (link)). Based on the manufacturer’s protocol, the library was constructed with an AmpliSeqTM Library PLUS for the Illumina kit (Illumina, USA). For each sample dataset, the mean sequence depth was above 1000x, and the 0.2x uniformity was not less than 0.85; otherwise, the library was reconstructed and sequenced. After library construction, sequencing was performed on an Illumina MiSeq Reagent Kit v3 (150 cycles) (Illumina, USA) DX system.

Bone marrow samples were collected before treatment, and tumor cells were purified by positive selection with anti-CD138 magnetic microbeads (Miltenyi Biotec, Germany). Genomic DNA was extracted from tumor cells using the QIAamp® Blood DNA Mini Kit (QIAGEN, Germany). The quantity of DNA was measured using a Qubit 2.0 fluorometer (Life Technologies, USA). Genotyping was performed using a sequencing panel of 387 genes (see Additional file 2 for details) by NGS technologies, and the average sequencing depth was 1000×. Genomic DNA (200 ng) was sheared with Enzyme Plus Library Prep Kit (iGenetech, China) and sequencing libraries were constructed using probes and TargetSeq One Kit (iGenetech, China) according to the instructions. Sequencing runs were performed on NovaSeq 6000 sequencing platform (Illumina, USA) using a NovaSeq 6000 S4 Reagent Kit v1.5 (300 cycles) (Illumina, USA). The raw sequencing data were available in the SRA database (https://www.ncbi.nlm.nih.gov/sra) under the accession number PRJNA826654.