2.4. Tagmentation-Mediated PCR (Tag-PCR)

Genomic DNA was extracted from CAR-T cells using a QIAamp DNA Blood Mini Kit (Qiagen, Hilden, Germany). We performed two-step measurement of DNA using Qubit 2.0 Fluorometer (Life Technologies, CA, USA) to ensure accuracy. In addition, we routinely measured A260/A280 ratio using NanoDrop (Life Technologies) to assess protein contamination and diluted target DNA to ensure the concentration of EDTA in DNA was <0.1 mM. Further, 50 ng DNA was applied to tagmentation reaction for 5 min at 58 °C using the transposase and transposon complex provided in Illumina Nextera DNA Library Preparation Kit (Illumina, San Diego, CA, USA). In this reaction, the DNA was fragmented at random positions and added with adapter sequences on both ends, serving as a scaffold for subsequent PCR reactions [4]. After purification with AMPure XP beads (Beckman Coulter, Brea, CA, USA), the tagmentation product was applied to the following two-step PCR.

In the first step of PCR, we performed 40-cycle PCR to amplify vector-genome junctions using PrimeSTAR GXL DNA Polymerase (TaKaRa Bio, Ohtsu, Japan), a Mastercycler pro S (Eppendorf, Hamburg, Germany), and primers, which were specific to an end of the transgene and adapter sequence, according to the manufacturers' instructions. We performed six patterns of PCR in parallel using 2 ng DNA for each reaction. The transgene-specific primer was tagged with an adapter sequence on its 5′ end, which served as a scaffold for the second step of PCR. In this step, platform-specific adapter sequences and individual indices for next-generation sequencer were attached to the first PCR product in the 14 cycles of thermal cycling. We used 1 μL of the first PCR product for the second step of PCR without any purification. The sequences of primers are listed in Supplementary Table 1, whereas paring of primers for the first PCR are summarized in Supplementary Table 2. In the second step of PCR, index numbers N723–N726, N716–N719, and N720–N722 were used for piggyBac, retrovirus, and lentivirus, respectively.

Lastly, the second PCR product was purified with AMPure XP beads, quantified, and subjected to sequencing on HiSeq2500 next sequencing platform with 2- × 150-bp paired end-reads (Illumina).

Three separate experiments using three different donors' PBMC were performed for each gene transfer system. After confirming that the profiles of integration sites did not significantly differ among the donors (Supplementary Fig. 2), we pooled the data for each gene transfer system.