Qiaamp blood mini kit

Genomic DNA was isolated from the tumor, NAT and PBMC single cell suspensions using QIAamp blood mini kits (Qiagen, Hilden, Germany). Targeted TRB-complementarity determining region 3 (TRB-CDR3) libraries were constructed using the ImmunoSEQ hsTCRB v3.0 kit (Adaptive Biotechnologies, Seattle, WA) on 1µg of genomic DNA isolated from the biospecimens according to the manufacturer’s protocol. Briefly, bias-controlled multiplex PCR was applied to amplify all possible rearranged genomic TCRβ sequences using an equimolar pool of the 45 TCR Vβ forward primers, and an equimolar pool of the TCR Jβ reverse primers. The following thermal cycling conditions were used for amplification: 1 cycle at 95°C for 15 minutes, 25 to 40 cycles at 94°C for 30 seconds, 59°C for 30 seconds, and 72°C for 1 minute, followed by 1 cycle at 72°C for 10 minutes (15 (link)). The final PCR products at 200bp length were pooled and sequenced at survey level resolution on the Illumina MiSeq platform (v3 150 cycle) in the Genomics Core Facility at the Fred Hutchinson Cancer Research Center as previously described (16 (link), 17 (link)).

Approximately 5 ml of Scope mouthwash was used to collect oral fluids and cellular materials. Samples were centrifuged at 8,000 g for 5 min to pellet cellular materials. The supernatant was transferred to another tube without disturbing the pellet. Pellets were stored at -80 ^oC prior to extraction. PBMC materials were matched with oral cells (same draw date) in every incidence except one intermittent shedder who had PBMC draw next day and for two never shedders who had PBMC materials within a month of the collection of oral cells. PBMC were isolated from blood collected in acid citrate dextrose (ACD) tubes by Ficoll (GE Healthcare) centrifugation with Leucosep tubes (Greiner bio-one). Red blood cells were lysed with ammonium-chloride-potassium (ACK) buffer following manufacturer’s protocol (Thermo Scientific). The purified PBMC were counted using a Nexcellom Cellometer Vision instrument. Pellets of approximately 1–2 million PBMC were obtained for each individual and stored at -80°C prior to DNA extraction.
Genomic DNA was extracted from oral cells and PBMC pellets using QIAamp Blood Mini Kits according to the manufacturer’s instructions (Qiagen). The quality and quantity of the DNA extracted were measured using a UV spectrophotometer at 260 and 280 nm (Nanodrop 2000). All DNA samples were stored at -80 ^oC.

In this report, we then tested the applicability of m-MCDA method using the spiked blood samples. The human blood samples were acquired from a healthy donor with the written informed consent. Our study was reviewed and approved by the ethics committee of the National Institute for Communicable Disease Control and Prevention, China CDC, according to the medical research regulations of the Ministry of Health China (Approval No. ICDC2014003).
A suspension of MRSA strain (S. aureus ATCC 43300) (1 × 10⁸ CFU ml^-1) was prepared in 1 ml of phosphate-buffered saline. The suspension was used for making a serial dilution (1 × 10⁷ CFU ml^-1, 1 × 10⁶ CFU ml^-1, 1 × 10⁵ CFU ml^-1, 1 × 10⁴ CFU ml^-1, 1 × 10³ CFU ml^-1, 1 × 10² CFU ml^-1, and 1 × 10¹ CFU ml^-1). Then, each dilution was centrifuged and re-suspended in 100 μl of blood sample (a 5-day negative blood culture). The DNA templates from spiked blood samples were extracted using DNA extraction kits (QIAamp Blood Mini Kits; Qiagen, Hilden, Germany) according to the manufacture’s instructions. The extracted genomic DNA was eluted in 100 μl of elution buffer and a volume of 1 μl of extracted templates was used for m-MCDA reactions. Non-contamination blood samples were used as NC. The experiments were carried out in duplicate to ensure reproducibility and accuracy.