Designstudio sequencing

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.

Panels for targeted sequencing covering the coding sequence (CDS) of the TP53 gene were designed online (Designstudio Sequencing, Illumina, USA) and shown in Supplemental Table 5. Library preparation was carried out with AmpliSeq^TM Library PLUS for Illumina, and sequencing was performed on a NextSeq^TM 550 system. Alignment and variant calling utilized the DNA Amplicon workflow (default parameters) on the BaseSpace Sequence Hub. The obtained variants underwent annotation with Annovar. Only exonic nonsynonymous or splice donor/acceptor site variants displaying a frequency below 0.001 in the gnomAD database were filtered for further analyses. The detailed procedures were described previously^{23 (link),54 (link)}.