Nextseq 550dx platform

Genetic mutations were detected by targeted NGS panels in 46 cases. Total DNA from PB, BM or LN samples was extracted for targeted NGS to detect gene mutations. Targeted NGS was also performed using the Ion Torrent/Illumina NextSeq 550Dx platform. The panels used are summarized in Supplementary Table 2. To minimize potential artifacts, only somatic variants with a PASS filter, read depth DP≥500 and VAF≥2% were retained. The filter criteria included 1) nonsynonymous mutations in the exonic region, splicing mutations, and UTR mutations in NOTCH1; 2) allele frequency of the mutated gene < 0.01 in the global population in the 1000G, ExAC, and gnomAD databases; and 3) other gene mutations that were proven to have clinical significance. Particular attention was given to mutated TP53, NOTCH1, MYD88, SF3B1, ATM, and MYC.

Genomic DNA and RNA samples were extracted from the same FFPE tissue sections using kit AllPrep DNA/RNA FFPE KIT (Cat No./ID:80234) according to the manufacturer’s protocols.
In this study, the preparation of both RNA and DNA sample library were performed according to the TSO500 Library Preparation Kit (Illumina, San Diego, CA, USA). Then, a two-step capture and enrichment of specific capture probes for DNA samples was performed, the DNA library and corresponding cDNA library of 8 samples was standardized by using the library homogenization method based on magnetic bead purification and sequenced using the Illumina NextSeq 550Dx platform. The Sequencing results were analyzed using TSO500 Docker pipeline.

The whole blood samples were collected from IE patients in the mean time with blood culture, and then about 300ul plasma (the whole blood sample centrifugated at 4°C) of each patient was used to extracted DNA by TIANamp Micro DNA Kit following the manufacturer’s instructions. DNA libraries were quality controlled using an Agilent 2100 Bioanalyzer (Agilent, USA) and an ABI StepOnePlus real-time polymerase chain reaction (PCR) system, and qualified libraries were sequenced on a NextSeq 550Dx platform (illumina, USA) using a 75 bp sequencing read length as previously described (Zhao et al., 2022 (link)). After the low-quality reads were removed, the clean reads were aligned to human genome to remove host sequence reads. All selected reads were mapped to the commercial pathogen database to annotate and analyze. Template free control (NTC) was obtained by repeated DNA extraction and sequencing using blank tubes filled with UltraPure Dnase/Rnase-free distilled water.
The data that support the findings of this study have been deposited into EMBL database with accession number PRJEB57970.

For patients enrolled, the following parameters were recorded carefully: age, sex, previous history, comorbidities [hypertension, type 2 diabetes, coronary artery disease (CAD), and chronic obstructive pulmonary diseases (COPD)], primary disease, use of invasive ventilator, stay time of intensive care unit (ICU), the reads of mNGS, and baseline laboratory examinations. In addition, the patients’ pre-hospital symptoms and patients’ CT imaging performance during hospitalization were carefully recorded. The clinical data of the patients were obtained through the electronic medical record system.
All patients had bronchoscopy performed, and about 5ml BALF was used to extracted DNA by TIANamp Micro DNA Kit following the manufacturer’s instructions. DNA libraries were constructed by DNA fragmentation, end-repair, adapter ligation, and PCR amplification, followed by sequencing. Agilent 2100 bioanalyzer (Agilent, USA) and ABI StepOnePlus Realtime PCR System were used for quality control of DNA library, the qualified libraries were sequenced on NextSeq 550Dx platform (illumina,USA) using 75bp sequencing read length. Positive control and negative control were included in all assays. The lower limit of detection of the assay was estimated to be <50 copies/ml (as per the instruction of the manufacturer). DNA integrity of the samples was confirmed by the presence of internal control DNA.

Genomic DNA extraction and library preparation with TruSight™ Oncology 500 (TSO 500) Library Preparation Kit (Illumina, San Diego, CA, United States) were performed following protocols described previously (Li et al., 2021 (link)). The library was sequenced on the Illumina NextSeq 550Dx platform with the paired-end run of 150 base pairs. The quality of sequencing data was verified by TSO 500 Docker pipeline. The process of SNVs and Indels mutation calling, TMB measuring, and reads filtering were performed following the description in the previous study (Li et al., 2021 (link)). Germline variants could be filtered out using an in-house built database, and all parameters were set according to the previous workflow (He et al., 2021 (link)).