Ampliseq library plus

Targeted high-throughput sequencing was applied for somatic alterations. A total of 57 genes were selected in this study (listed in Table S2). Most genes were frequently altered in DLBCL, according to data from several previously published large-scale DLBCL group studies (32 (link)–34 (link)). Using genome build hg19/GRCh37 as a reference, a sequencing panel covering the coding sequences within five intronic base pairs around exons in 57 genes was designed online (Designstudio Sequencing, Illumina, San Diego, USA). Sequencing libraries were prepared with AmpliSeq™ Library PLUS for Illumina, using 20 ng of input genomic DNA per sample. Library sequencing was performed to 2000X coverage on a NextSeq™ 550 system using an Illumina NextSeq™ 500/550 High Output v2 Kit (Illumina, San Diego, USA). The alignment and variant calling were performed using a DNA Amplicon workflow with default parameters on BaseSpace Sequence Hub (Illumina). The generated variants were further annotated using ANNOVAR (35 (link)). Further details are described in the Supplementary Methods.

The NGS libraries were prepared according to the AmpliSeq for Illumina On-Demand, Custom and Community Panels Reference Guide Protocol, DNA Panels Standard Workflow Procedure for Three Primer Pools, using reagents from the AmpliSeq™ Library PLUS for Illumina®. The concentration of the input DNA was measured using the Qubit dsDNA BR Assay and the concentration of the constructed libraries was assessed using the Qubit dsDNA HS Assay on the Qubit 2.0 Fluorometer (Thermo Fisher Scientific) following the manufacturer’s protocol. The quality of the constructed libraries was assessed using the DNA Hi Sens Lab Chip (PerkinElmer, Waltham, USA) according to the DNA High Sensitivity Assay User Guide for LabChip GX Touch/GXII Touch Standard Sample Workflow. Libraries were denatured and diluted according to the MiSeq System Denature and Dilute Libraries Guide Protocol A: Standard Normalization Method for MiSeq Reagent Kit v2, with a PhiX library used as a sequencing control. Pooled sequencing of 6 samples per run was conducted on an Illumina Miseq using the MiSeq Reagent Micro Kit v2 (2 × 150 base pair (bp), paired end) to a minimum coverage depth of 5000 × according to the manufacturer’s protocol.

DNA from 4–8 sections (4–5 μm each) of formalin-fixed paraffin-embedded (FFPE) tumor samples (which were selected by a pathologist as described above) was extracted using the “QIAamp DNA FFPE GeneRead Kit” (Catalogue Reference: 180134; Qiagen, Germantown, MD, USA). Subsequently, the whole exonic region of IDH1 and IDH2 genes was then sequenced using an Illumina MiSeq device (version 3.1.0.13) following the manufacturer’s instructions, as previously described [39 (link)]. In brief, after DNA quantification from FFPE tumor samples using Qubit 1× dsDNA HS Assay Kit (Catalogue Reference: Q33230; Thermo Fisher Scientific, Waltham, MA, USA), 50–150 ng was used for mutational analysis by AmpliSeq methodology (Illumina, Inc., San Diego, CA, USA). AmpliSeq Library PLUS was used for library preparation (Catalogue Reference: 20019101; Illumina, Inc., USA), followed by the amplification of target regions and second amplification of libraries, which were diluted and denatured for bridge clonal amplification and paired-end sequencing using MiSeq Reagent kit v2 (300-cycles) (Catalogue Reference: MS-102-2002; Illumina, Inc., USA) in a MiSeq instrument (Illumina, Inc., USA). Variant calling files annotation, and the identification and classification of detected genetic variants were performed with the VariantStudio software v3.0 (Illumina, Inc., USA).