Na12878

Off-target sequencing reads from the tumor-sequencing data were used to generate genome-wide copy number data for 29 lowest quintile and 25 highest quintile breast cancers using CopywriteR^{38 (link)}, utilizing a normal lymphocyte DNA control run in the same sequencing batch for the normalization baseline (NA12878, Coriell Institute). Data were imported into NEXUS Copy Number^TM (software v8.0 with build version 9169, BioDiscovery Inc.), segmented using a FASST2 segmentation algorithm, and visualized. Comparisons between groups were made using Nexus applying thresholds of p < 0.05 and at least 25% frequency difference. Within the Nexus software package gains were defined as log₂ ratio >0.3 and losses at <−0.3. High copy number gains were called if the log₂ ratio was >1.2 and homozygous deletions <−1.2.

We estimated the quantitative precision of the MGISEQ-2000 sequencer. The sequencing data were generated by the dilution samples, which were produced by mixing the universal methylated DNA standards (Zymo, D5014-2) and the gold standard reference samples NA12878 (Coriell) at the predefined ratios of 0.002, 0.01, 0.02 and 0.05 (five replicates per ratio). The mock dilution samples (water) were defined as 0 (four replicates). Then, we evaluated the quantitative precision using the expected spike-in ratios compared to the expected dilution ratios. The estimated spike-in ratio of the dilution sample was calculated as the mode of estimated fractions of all target regions in the dilution sample. $$F_{i,j}=\left({\text{AMF}}_{i,j}-{\mu }_i\right)/\left(1-{\mu }_i\right)$$ $${\mu }_i={\sum }_{k=1}^N{\text{AMF}}_{i,k}/N$$ where i represented the index of the targeted regions, k represented the index of baseline sample, j represented the index of dilution samples, N represented the count of the baseline samples.

For technical validation, the reference standard NA12878 (Coriell Institute, USA) was obtained, as a purified DNA sample, from a woman who was a participant in the International HapMap Project [31 ]. Three iterations of WES and WGS sequencing experiments were performed from the single aliquot of RS NA12878 extracted DNA, thus representing technical replicates of the same biological sample.
The blood–saliva paired samples were collected from each of the 10 participants (five healthy males and five healthy females with no clinical suspicion of any disease, aged 16 to 62 years at the time of sample collection) on the same day. The venous blood samples (4 mL/participant) were collected into Vacuette K2EDTA tubes (Becton Dickinson, USA), tempered for 30 min to room temperature, and then stored at 4 °C. To minimize the ratio of non-human DNA contamination in the saliva samples, the saliva collection was strictly controlled. Participants washed their teeth by toothbrush without toothpaste for approx. 1 min., rinsed their mouths with water, and avoided eating or drinking for 30 min before collecting the sample. A total of 2 × 1 ml of saliva was collected from each participant into 4 mL of PBS pH 7.2 (HiMedia, Germany) including PSA (penicillin 100 IU/ml, streptomycin 100 µg/ml, amphotericin B 2.5 µg/ml, sodium deoxycholate 2.5 µg/ml; Serana, Germany) and stored at 4 °C.

About 0.5–1 µg of Lambda or 3–5 µg of purified NA12878 (Coriell Institute) fragments were used as input for sequencing library preparation. A dA-tailing reaction was performed in the presence of 1 mM dATPs and 1X Thermopol (NEB) with 5U of Taq DNA Polymerase 72 °C for 5 m. The manufacturer-recommended amounts of sequencing adapters and ligation buffer from the SQK-LSK109 kit (Oxford Nanopore) were added to this reaction along with NEBNext Quick T4DNA Ligase. The ligation reaction was carried on at room temperature for 15 min before AMPURE XP beads were cleaned up. The library was quantified with a Qubit fluorometer before mixing with sequencing buffer loading beads and loading on a primed flow cell. Priming was carried out according to the manufacturer’s recommendations. Nanopore Sequencing was carried out on a MinION sequencer using FLO-FLG001 flongles or Minion R9.4 flow cells from ONT. The experiment was set up and run with live base calling performed using MinKNOW software with default configurations.

Primers were designed to bind in the intronic regions flanking the retrotransposon insertion (MECP2-int4-gDNA-Fwd: 5′-GCCTCTCCAAAGTTCAGCAAC-3′; MECP2-int4-gDNA-Rev: 5′-TGCCCTGAGTGGGAAGTTCT-3′). PCR was performed using PrimeStarGXL DNA Polymerase (Takara Bio) according to manufacturer’s instructions. Then, 50 ng genomic DNA from the proband (A0131084), both parents (A0131084-M and A0131084-P) and NA12878 (Coriell) was amplified, alongside a No Template Control reaction. Reactions were run on a 1% E-Gel EX (Thermo Fisher) and visualized using a GelDoc XR (Bio-Rad).