X10 platform

DNA samples were extracted from patient PBMC or pretreatment and post-treatment tumor biopsies from patient 5 using a DNA extraction kit (Qiagen, USA), followed by library construction with two rounds of PCR-based amplification. CDR3 fragments were first amplified using specific primers for each V and J gene, and target fragments of multiplex-PCR products were purified using magnetic beads (A63882, Beckman, Germany). Next, PCR was performed using universal primers, and target fragments 200–350 bp were retrieved and purified by QIAquick Gel Purification Kit (Qiagen, USA). PCR products were then sequenced using the Illumina X10 platform. Single-read CDR3 sequences were eliminated, and the remaining sequences were analyzed to evaluate TCRVβ IMGT clonality of patients before and after treatment, as previously described.53 (link)

Tumor tissue was collected, snap-frozen, and stored in liquid nitrogen or embedded in paraffin. Blood samples from the corresponding patient were used as the control. Genomic DNA was extracted from these samples following the protocol described in a previous manuscript (13 (link)). Targeted sequencing was performed using multiple gene panels (Genetron Health Co. Ltd., Country), and the Illumina X10 platform was used for sequencing.

The WES sequencing protocol prescribed by Giannakis et al. [17 (link)] was used in our study. In terms of
models, the tumor tissues preserved in the liquid nitrogen were centrifuged with 1 min, the centrifugal rate of 9,400 ×g/min. For the patient, the
formalin-fixed, paraffin-embedded tumor tissues were sectioned into 5 µm for sequencing, and the normal tissues adjacent to tumor were regarded
as control. QIAGEN DNA kit (QIAGEN NV, Hilden, German) was used to extract genomic DNA, and the Quant-iT Pico Green dsDNA Assay Kit (Invitrogen/ThermoFisher
Scientific) was conducted to check the DNA quality. The whole-exome capture libraries (final concentration >20 ng/µl) were constructed by
shearing, end repair, phosphorylation and ligation to barcoded sequencing adaptors. SureSelectXT Human All Exon V6 (Agilent Technologies) was used to capture
DNA and the Illumina X10 platform (Illumina Inc., San Diego, CA, USA) was then used to sequenced the samples. The method of MuTect [18 (link)] was performed to detect the somatic mutations, and the somatic cell insertion and deletion markers were detected by the method of
Indelocator and Strelka [19 (link)]. The mutation analysis of WES data was performed using human genome build hg19 as the
reference genome.