Ampure xp beads

Genomic DNAs from FFPE samples and whole blood control samples were extracted with QIAamp DNA FFPE Tissue Kit and DNeasy Blood and tissue kit (Qiagen), respectively, and quantified by Qubit 3.0 using the dsDNA HS Assay Kit (ThermoFisher Scientific). Library preparations were performed with KAPA Hyper Prep Kit (KAPA Biosystems). A customized panel targeting 425 cancer‐relevant genes was used for hybridization enrichment (Appendix [Link], [Link]). The capture reaction was performed with Dynabeads M‐270 (Life Technologies) and xGen Lockdown hybridization and wash kit (Integrated DNA Technologies) according to manufacturers’ protocols. Captured libraries were on‐beads PCR amplified with Illumina p5 (5’ AAT GAT ACG GCG ACC ACC GA 3’) and p7 primers (5’ CAA GCA GAA GAC GGC ATA CGA GAT 3’) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by qPCR using KAPA Library Quantification kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target‐enriched library was then sequenced on HiSeq4000 NGS platforms (Illumina) according to the manufacturer's instructions.

Genomic DNA from FFPE sections or biopsy samples and whole blood control samples were extracted using the QIAamp DNA FFPE Tissue kit and DNeasy Blood and Tissue Kit (Qiagen, USA), respectively. Sequencing libraries were prepared using the KAPA Hyper Prep Kit (KAPA Biosystems) according to the manufacturer’s instructions for different sample types. Customized xGen Lockdown Probes (Integrated DNA Technologies) targeting 437 cancer-related genes were used for hybridization enrichment. The capture reaction was performed with Dynabeads M-270 (Life Technologies) and xGen Lockdown Hybridization and Wash Kit (Integrated DNA Technologies) according to the manufacturer’s protocols. Captured libraries were constructed by on-beads PCR amplification with Illumina p5 (5’-AAT GAT ACG GCG ACC ACC GA-3’) and p7 primers (5’-CAA GCA GAA GAC GGC ATA CGA GAT-3’) on the KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification using Agencourt AMPure XP beads. Libraries were quantified by quantitative PCR using the KAPA Library Quantification Kit (KAPA Biosystems). Library fragment size was determined by Bioanalyzer 2100 (Agilent Technologies). The target-enriched library was then sequenced on the HiSeq4000 NGS platform (Illumina) according to the manufacturer’s instructions. The mean coverage depth was 317X for the whole blood control samples and 1320X for the tumor samples.

Targeted sequencing covering exons and selected introns of leukemia‐ and lymphoma‐related genes was peformed for 20 patients with PB‐DLBCL patients from two of these institutes. Formalin‐fixed, paraffin‐embedded tumor tissues were used for genomic DNA extraction with a QIAamp DNA FFPE Tissue Kit (Qiagen), and the paired normal control DNA of peripheral blood mononuclear cells (PBMCs) was extracted with a DNeasy Blood & Tissue kit (Qiagen) following the manufacturer's instructions. Tumor genomic DNA and matched PBMCs were fragmented into 300–350 bp fragments using a Covaris M220 instrument (Covaris). Sequencing libraries were prepared with a KAPA Hyper Prep kit (KAPA Biosystems) with optimized protocols. Libraries were then subjected to PCR amplification and purification before targeted enrichment. The probes for targeted sequencing panel covered exons and selected introns of leukemia‐ and lymphoma‐related genes and were produced by Nanjing Shihe Jiyin Biotechnology Inc. (Nanjing, China). Afterward, the samples were purified by Agencourt AMPure XP beads, quantified by a KAPA Library Quantification kit (KAPA Biosystems), and sized with an Agilent Technologies 2100 Bioanalyzer (Agilent Technologies). Finally, the enriched libraries were sequenced on HiSeq 4000 NGS platforms (Illumina) to coverage depths of 1500× after removing PCR duplicates for FFPE.

Libraries with different indexes were pooled together in optimized ratios to reach up to 2 μg of total DNA. Human Cot-1 DNA (Life Technologies) and xGen universal blocking oligos (Integrated DNA Technologies) were added as blocking reagents to reduce non-specific hybridization. Customized xGen lockdown probes (Integrated DNA Technologies) targeting 5,804 exons of 382 cancer-relevant genes and 37 introns of 16 fusion genes were used for hybridization enrichment (Supplementary Table 1). The capture reaction was performed with NimbleGen SeqCap EZ Hybridization and Wash Kit (Roche) and Dynabeads M-270 (Life Technologies) according to manufacturers’ protocols. Post-captured libraries were PCR amplified with Illumina p5 (5′ AAT GAT ACG GCG ACC ACC GA 3′) and p7 primers (5′ CAA GCA GAA GAC GGC ATA CGA GAT 3′) in KAPA HiFi HotStart ReadyMix (KAPA Biosystems), followed by purification with Agencourt AMPure XP beads, and quantified by qPCR method using KAPA Library Quantification kit (KAPA Biosystems). The size distribution of the library was analyzed by Bioanalyzer 2100 (Agilent Technologies). Target-enriched libraries were then sequenced on Illumina MiSeq or HiSeq4000 NGS platforms (Illumina) according to manufacturer's instructions. Targeted capture and sequencing performance of all the samples were summarized in Supplementary Table 3.