Magmax cell free dna isolation kit

Peripheral blood samples were collected into tubes containing EDTA and centrifuged (2000×g, 10 min, 4 °C) within 4 h of collection. To prevent cellular DNA contamination, the plasma supernatants were carefully removed and recentrifuged (16,000×g; 10 min at 4 °C). The prepared plasma samples were stored at −80 °C until further analysis.
ccfDNA was extracted using the MagMAX™ Cell-Free DNA Isolation Kit (Applied Biosystems, Carlsbad, CA, USA) following the manufacturer’s instructions. The isolated ccfDNA was eluted in 20 μl of the provided solution and stored at −20 °C prior to quantitative and qualitative analyses.

The genomic DNA isolated from amniocytes was fragmented into an average size of 250 bp, whereas cell‐free DNA (cfDNA) was isolated with MagMAX™ Cell‐Free DNA Isolation Kit (Applied Biosystems™ cat.: A29319) according to the manufacture's instruction. Library construction, quality control, and sequencing were performed as previously described (Liang et al., 2013; Qi et al., 2018; Song et al., 2013). Briefly, 2.5 ng of cfDNA or fragmented DNA was used for the preparation of sequencing libraries. The 8‐bp barcoded sequencing adaptors were ligated to fragments and amplified by PCR. Purified libraries were sequenced using NextSeq 550AR (Annoroad Gene Technology Co., Ltd China). For the confirmatory tests, an average of 7.5‐M reads with 40 bp in length was generated for each amniocytes sample, accounting for 0.1× of the human genome; for each maternal plasma sample, an average of 4.2‐M reads with 40 bp in length and Q30 > 95% was generated for further analysis. Samples that failed to pass the in‐house quality control of cfDNA testing in the first round were subjected to repeat testing. In total, 20,232 samples had successful NIPSCCD results after the second test. For the evaluation of the performance of NIPSCCD, 20,003 successfully tested samples with confirmatory results or follow‐up results were used.

Plasma samples were collected using PAXgene Blood ccfDNA tubes (#768165; Qiagen, Hilden, Germany). Blood samples were collected weekly during hospitalization following transplantation, and then every time the patients underwent medical examinations or transbronchial biopsies. A total of 372 plasma samples were obtained from 30 patients (average, 12,4/patient). Plasma was separated by centrifugation (2,000 ×g, 15 min, 18°C) and stored at −80°C in the Teseo Biobank of the Department of Medical Sciences of University of Turin (http://www.progettoeccellenzateseo.unito.it/) until further processing. Cell-free DNA (cfDNA) was extracted from 1 ml of plasma using MagMAX Cell free DNA isolation kit (#A29319, Applied Biosystems, Waltham, MA, United States) and stored at −20°C until analysis.

CfDNA was isolated using the MagMAX Cell-Free DNA Isolation Kit (Applied Biosystems) from these plasma samples. The concentration and size distribution of cfDNA were measured by Qubit (Invitrogen) and BioAnalyzer (Agilent), respectively. Samples were randomly distributed into different batches for library preparation and sequencing. Cases and their matched controls were always generated in the same batch. Library construction was performed on 1ng of cfDNA using the KAPA HyperPrep Kit (Roche) and NEXTFLEX Unique Dual Index Barcodes (300nM final concentration, PerkinElmer). Libraries were sequenced on Illumina NovaSeq 6000 in PE150 mode. The cfDNA WGS data generated here was processed in the same way as we did for the public cfDNA WGS dataset. The z-score-transformed IFS signals were extracted at the hotspots, which were identified in the public dataset from the same cancer types and their controls. The normalized IFS signals were finally utilized for the performance validation of the machine learning models.

Peripheral venous blood samples (n = 115) were collected for patients at three time points: two days before surgery (pre-op), 10 days after surgery (post-op), and at the end day of the last chemotherapy (post-chemo). At least 20 mL of blood was taken into EDTA-containing tubes. Plasma was separated within 4 h after sample collection. Obtained plasma was centrifuged at 2000 g for 5 min and at 16,000 g for 10 min, immediately aliquoted, and stored at −80 °C. cfDNA was isolated from 4 mL of plasma using a MagMAX Cell-Free DNA Isolation Kit (Applied Biosystems, Foster City, CA, USA) with a KingFisher Duo Prime Magnetic Particle Processor (Thermo Fisher Scientific, Waltham, MA, USA), according to each manufacturer’s instructions. The concentration of the purified plasma cfDNA was measured using a Qubit 2.0 fluorometer (Thermo Fisher Scientific, Waltham, MA, USA) in combination with a Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA) according to the manufacturer’s instructions.