Qubit uorometer

5hmC libraries were prepared following a previously described selective chemical labeling technique [17] .
Brie y, 10 ng cfDNA spiked with control oligos (0.01 pg of unmodi ed, methylated, or hydroxymethylated oligos per 10 ng cfDNA) was end repaired, 3'-adenylated, ligated to DNA Barcodes (Illumina Compatible), labeled with 5hmC and pulled down, and ampli ed with 14 cycles of PCR ampli cation using KAPA Hyper Prep Kit (Kapa Biosystems) according to the manufacturer's instructions. The PCR products were puri ed using 1X AMPure XP beads according to the manufacturer's instructions. The DNA concentration of each library was measured with a Qubit uorometer (Life Technologies), and sequencing was performed on the Illumina NextSeq 500 platform.

Peripheral blood (5-10 ml) was collected in EDTA anticoagulant tubes (BD). The samples were placed on ice and sent to the lab within 2 hours. Plasma samples were collected from peripheral blood after centrifugation for 10 min at 1600 g (4°C) and for 10 min at 16,000 g (4°C). The prepared plasma samples were immediately stored at -80°C until puri cation. cfDNA was extracted from 3-5 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) according the manufacturer's protocol. Concentrations and quality of cfDNA were quanti ed by Qubit uorometer (Life Technologies) and 2% agarose gel electrophoresis (Invitrogen).

5hmC libraries were prepared following a previously described selective chemical labeling technique [17] .
Brie y, 10 ng cfDNA spiked with control oligos (0.01 pg of unmodi ed, methylated, or hydroxymethylated oligos per 10 ng cfDNA) was end repaired, 3'-adenylated, ligated to DNA Barcodes (Illumina Compatible), labeled with 5hmC and pulled down, and ampli ed with 14 cycles of PCR ampli cation using KAPA Hyper Prep Kit (Kapa Biosystems) according to the manufacturer's instructions. The PCR products were puri ed using 1X AMPure XP beads according to the manufacturer's instructions. The DNA concentration of each library was measured with a Qubit uorometer (Life Technologies), and sequencing was performed on the Illumina NextSeq 500 platform.

Peripheral blood (5-10 ml) was collected in EDTA anticoagulant tubes (BD). The samples were placed on ice and sent to the lab within 2 hours. Plasma samples were collected from peripheral blood after centrifugation for 10 min at 1600 g (4°C) and for 10 min at 16,000 g (4°C). The prepared plasma samples were immediately stored at -80°C until puri cation. cfDNA was extracted from 3-5 ml of plasma using the QIAamp Circulating Nucleic Acid Kit (Qiagen) according the manufacturer's protocol. Concentrations and quality of cfDNA were quanti ed by Qubit uorometer (Life Technologies) and 2% agarose gel electrophoresis (Invitrogen).

Whole genomic DNA was extracted from peripheral blood of the 15 family members using DNA Isolation Kit (Bioteke, AU1802) as previously described [13] . Concentrations of each DNA sample were measured on a Qubit uorometer (Invitrogen, Q33216) using Qubit dsDNA HS Assay Kit (Invitrogen, Q32851).
Meanwhile, 1% agarose gel electrophoresis was performed for quality control of each DNA sample.

Genomic DNA was extracted using a DNeasy UltraClean DNA isolation kit according to the manufacturer's instructions (Qiagen, Hilden, Germany). DNA was quanti ed using a Qubit ® uorometer (Invitrogen, CA, USA) high-sensitivity assay, before dilution to the required concentration in RNase-free water and puri cation on AMPure XP beads (Beckman Coulter). Sequencing libraries were prepared from 0.5ng/µl of RNA free genomic DNA. A total of 282 isolates were included for genomic sequencing using the Nextera-XT DNA sample preparation kit (Illumina) and whole-genome sequencing performed using the Illumina NextSeq sequencing platform, generating paired-end reads (2 x 150bp). Reads were uploaded to the Sequence Read Archive under Bioproject ID PRJNA543206.
Paired-end reads were quality-assessed and trimmed using FastQC and Trimmomatic as described above [19, 20] . Trimmed reads were assembled into scaffolds using SPAdes version 3.13.1 [56] . Scaffolds shorter than 500 bp were discarded from analysis. Genome contamination and completeness was assessed using CheckM version 1.0.13 [47] . To con rm assembly quality, only genomes conforming to all the following criteria were included in further analysis: (i) scaffold N50 of >20 kbp (ii) 90% of assembled bases at > 5x read coverage (iii) completeness of > 95% (iv) contamination of < 5% (v) complete 16S rRNA gene sequence.