Le220

Up to 300 ng of purified DNA were fragmented to ~200 bp via Covaris LE220 using previously optimised shearing conditions for FFPE inputs. Library fragments were prepared via KAPA HyperPrep using Agilent SureSelect XT HS adaptors and pre-capture primers. Exome capture was performed as per Agilent SureSelect XT HS manufacturer protocols using the Agilent Clinical Research Exome V2 bait panel. Final libraries were sequenced on NextSeq500 in paired-end 75 bp configuration to a mean coverage of 80× for PBMC germline samples and 150× for FFPE tumour samples. Samples were demultiplexed and converted to fastq using bcl2fastq2 (version 2.20) and analysed using the nf–core Sarek-v3.1.2 pipeline [30 (link)] against the GRCh38 human reference genome (Ensembl). Briefly, initial alignment was performed using BWA-MEM v0.7.17 [31 ], and base-score recalibration was conducted using the GATK4 best-practices workflow with GATK4.4.3 [32 (link)]. Variant calling was also conducted using the Sarek v3.1.2 pipeline using GATK-Mutect2, and snpEff v5.1 [33 (link)] was used to characterise the variants.

For the next-generation DNA sequencing of rice, total genomic DNA was prepared from young leaves rice using the CTAB (cetyl trimethylammonium bromide) method (Murray and Thompson 1980 (link)). The genomic DNA was fragmented by Covaris LE220. Sequencing libraries were constructed according to the manufacturer’s instructions with an Illumina Genomic DNA Sample Preparation Kit. Short read sequences (76 bases) were generated by Illumina Genome Analyzer IIx with TruSeq SSB Kit v5-GA.

The microbial DNA was sheared into small fragments using a mechanical method by ultrasonication (Covaris, LE220, Massachusetts, USA). We normalized 100 ng of DNA into 52.5 µL and transferred it to Covaris microtubes. The DNA was sheared using the following setting: Duty Factor 20%, Intensity 5, Peak/Displayed Power 450 W, and cycle/Burst 200, with a fragment size of 350 bp. The libraries were prepared using Nano DNA Library preparation (Illumina, San Diego, CA, USA) according to the manufacturer’s protocol, and each sample was labeled using IDT-ILMN TruSeq DNA UD Indexes (96 Indexes). The library quality was checked by running the samples in an Agilent 2100 bioanalyzer (Agilent Technologies, Waldbronn, Germany) using a high-sensitivity DNA chip (Agilent Technologies, Germany). The libraries were quantified using a Qubit 4 fluorometer (Invitrogen, Thermo Scientific, Singapore), and the concentration was calculated in nM. Next, MiSeq was used to sequence the libraries. A total of 25 nM from each sample was pooled, and the final concentration of the pooled libraries was measured using Qubit 4. The library denaturation and the PhiX control denaturation were done according to the manufacturer’s protocol. The final sample of 600 µL (10 pM) with 5% PhiX control was loaded to the MiSeq (Illumina, San Diego, USA) for sequencing.

DNA from blood was extracted using the QIAGEN QIAamp Circulating Nucleic Acid Kit (Qiagen). Genomic DNA from PBMCs was extracted using the DNeasy Blood and Tissue Kit (Qiagen). After extraction, genomic DNA was sheared to resemble cfDNA using an ultrasonicator (LE220, Covaris). Library preparation for TS, sWGS, and cfMeDIP was performed only for samples with >40 ng DNA yield. TS was excluded for samples with <40 ng DNA yield. Samples with <10 ng DNA yield were not processed.

Four blood spots with the diameter of no less than 8 mm were gathered from DBSs. Apart from that, the genomic DNA extraction system kit (QIAamp DNA Blood Midi Kit, Qiagen, Germany) was adopted, while DNA concentration was 3–25 ng/μl, and DNA purity (OD 260/280) reached 1.8–2.0. The genomic DNA is broken into small DNA fragments with a main band of 100–500 bp by Covaris LE220 ultrasonic instrument (Massachusetts, USA), and then the broken DNA fragments are screened by magnetic beads. The size of the screened main fragment is 150 bp–200 bp. The CH-related genes DUOX2, DUOXA2, TSHR, NKX2-1, NKX2-5, FOXE1, PAX8, GLIS3, TG, TPO, SLC5A5, SLC26A4andIYDwere selected by a gene capture strategy, using Agilent 2100 Bio analyzer and BMG following the manufacturer's protocol. The high-throughput sequencing of the qualified enriched libraries was performed on MEGISEQ-2000 sequencer (BGI, China).

HiPure Tissue DNA Mini Kit (Magen) was used for tissue genomic DNA extraction. After quantification by Qubit fluorometer, 1% unmethylated Lambda DNA was added to 200 ng of gDNA, and then randomly fragmented to 300-bps insert size with Covaris LE220. After end repair and adenylation, methylated adapters were ligated to the fragmented DNA. Bisulfite treatment was performed according to the EZ DNA Methylation-Gold kit (Zymo Research) instruction manual. KAPA HiFi HotStart Uracil + ReadyMix (2×) was used to amplify and purify the DNA fragments. Next, the Qubit Fluorometer dsDNA HS Assay (Thermo Fisher Scientific) and Agilent BioAnalyzer (Agilent) were used to measure and analyze the size distribution of the sequencing library; 2×150 paired-end reads sequencing is performed using an Illumina NovaSeq6000 following Illumina-provided protocols at Mingma Technologies Co., Ltd.