Nextseq 550Dx Single-end Sequencing

Libraries were constructed for the DNA and cDNA samples using a Nextera XT DNA Library Prep Kit (Illumina, San Diego, CA) (17 (link)). The initial input of DNA is 5–100 ng. Firstly, DNA needs to be fragmented to obtain 150–250 bp inserts, followed by terminal repair and adapter connection, and finally, library amplification to construct a library that meets the requirements of sequencing. Library was quality assessed by Qubit dsDNA HS Assay kit followed by High Sensitivity DNA kit (Agilent) on an Agilent 2100 Bioanalyzer. Library pools were then loaded onto an Illumina Nextseq 550Dx sequencer for 75 cycles of single-end sequencing to generate ~20 million reads for each library. For negative controls, we also prepared PBMC samples with 10⁵ cells/mL from healthy donors in parallel with each batch, using the same protocol, and sterile deionized water was extracted alongside the specimens to serve as non-template controls (NTC).