Nextseq 550dx

A micro-sample genomic DNA extraction kit (DP316, Tiangen) was used to extract the nucleic acid. NEBNext Ultra II DNA Library Prep Kit (New England Biolabs Inc.) was used to construct Illumina sequencing libraries and Nextseq 550 DX (75 bp single-end reads; Illumina) was used for sequencing. An alignment tool (Burrows-Wheeler Alignment) was used to map to a human reference genome (GRCh38) to exclude human sequence data. The remaining sequencing data were aligned to NCBI nt database by SNAP. The specific detection method of mNGS can be referred to our previous report (Zhang et al., 2022 (link)).

Genomic DNA was extracted from peripheral blood using a QIAsymphony DNA Mini Kit (Qiagen, Hilden, Germany). A custom capture panel (Dxome, Seoul, Republic of Korea) targeting coding exons and intron-exon boundaries of 497 genes related to hematologic disorders (Supplementary Table S1) was used. Prepared libraries were hybridized with capture probes and sequenced as paired-end reads (2 × 150 bp) using NextSeq 550Dx (Illumina, San Diego, CA, USA). NGS data analysis was performed with DxSeq Analyzer (Dxome). Single-nucleotide variants, small insertion and deletions, and copy number variants were identified [13 (link), 14 (link)]. All variants were classified and reported as a 5-tier system according to the American College of Medical Genetics and Genomics and the Association for Molecular Pathology guidelines [15 (link)]. Genes included in our panel that are associated with HHAs are classified according to their relevant phenotypes in Table 1.
Table 1

Genes included in our panel classified according to phenotype

Phenotype	Genes included in the panel
Membranopathy	ANK1, SPTB, SPTA1, SLC4A1, EPB42, EPB41, PIEZO1, RHAG
Enzymopathy	G6PD, PKLR, AK1, AK2, ALAS2, GPI, NT5C3A, GCLC, GPX1, GSR, GSS, HK1, BPGM, TPI1, PFKL, PFKM, PGK1, ALDOA
Hemoglobinopathy	HBB, HBA1, HBA2, HBD

The qualified DNA libraries were subjected to sequencing on the NextSeq 550Dx platform (Illumina), employing a sequencing read length of 75-bp.

Based on high-throughput sequencing technology, the DNA sequence of the microorganism in the sample was obtained and compared with the microbial gene bank to identify pathogenic microorganisms. The detection process includes nucleic acid extraction, library construction, computer sequencing, bioinformatics analysis, and report interpretation.^{[16 (link)]} The detection process was based on Illumina NextSeq550Dx (Illumina) sequencing systems.

Targeted next-generation sequencing was performed for the tumor-only samples of the enrolled cases using AMC OncoPanel version 4.2, as described previously [11 (link)]. Briefly, the tumor area of each sample was bluntly dissected from sections of the tumor tissue blocks and the DNA was extracted and assessed for its quality. Subsequently, the samples that were appropriate for further analysis were subjected to sequencing using NextSeq 550Dx (Illumina Inc, San Diego, CA). After these bioinformatics analyses, all identified variants were manually reviewed by an experienced pathologist (H.S.H.) using an IGV genomic viewer system [16 (link)] to remove false-positive variants. All output files were integrated and visualized using R package maftools version 2.6.05 [17 (link)].

Genomic DNA and RNA was extracted from 5 μm sections of formalin fixed paraffin embedded tissue (FFPE) sections mounted on slides using the QIAamp DNA FFPE Tissue Kit (Qiagen, Hilden, Germany), RNeasy FFPE Tissue Kit (Qiagen), or AllPrep DNA/RNA FFPE Kit (Qiagen). DNA was set aside for methylation analysis as described below. Next-generation sequencing (NGS) was performed using commercial TruSeq RNA Exome panel (Illumina). Exome RNA sequencing libraries were prepared with 100 ng tumor RNA using the Illumina RNA Prep with Enrichment (L) Tagmentation kit (Illumina) with Exome Probe Panel (Illumina). Final enriched libraries are sequenced on NextSeq 550DX or NovaSeq 6000 (Illumina).