Qiacube platform

The commercially available Seeplex DV mRT-PCR assay (Seegene, Seoul, Korea) was used to simultaneously detect group A rotaviruses, AdV 40 and 41 (species F), noroviruses GI and GII, and astroviruses [10 (link), 11 ]. Nucleic acids were extracted from fecal suspensions by using QIAamp DNA Mini kit (Qiagen, Hilden, Germany) and QIAcube platform (Qiagen). The nucleic acid was amplified using a PTC-200 thermal cycler (Bio-Rad, Hercules, CA, USA), and the PCR products were visualized after electrophoresis on an agarose gel. All procedures were performed according to the manufacturers' instructions.

The wet lab methodology for the generation of the training dataset is described
elsewhere.^{14 (link)} For the novel test cohort, briefly, formalin-fixed
paraffin embedded (FFPE) tumor tissue slides were reviewed for tumor purity by a
licensed molecular pathologist and tumor-rich regions were macrodissected. DNA
extraction was performed using AllPrep kits on an automated Qiacube platform
(Qiagen). Paired germline DNA was extracted from an EDTA blood collection tube
on an automated Qiasymphony system. Library preparation was performed using KAPA
HyperPlus library preparation kits (Roche) and whole exome hybrid capture was
performed using IDT xGen Research Panel v1 kits. Samples were pooled and
sequenced on an Illumina HiSeq 2500 or HiSeq 4000 instrument. Sequencing was
performed under CLIA/CAP guidelines in the Providence clinical genomics
laboratory.

This study was performed retrospectively by using stored specimens. The specimens were collected in Dongtan Sacred Heart Hospital, Korea in April 2014, and from December 2014 to April 2015. During these periods, respiratory specimens, sputum, or nasopharyngeal swabs, and any nucleic acids from the specimens that showed positive results according to the Anyplex II RV16 detection kit were collected and stored at −70℃ until further analysis. Stored specimens underwent further evaluations, as explained in the following sections. When the stored specimens were unavailable or the quantity was too small, then the stored nucleic acids were used for the experiments.
During the collection periods, 248 stored respiratory specimens and 6 stored nucleic acids were used. The six nucleic acids were used because the frequency of coronavirus 229E and parainfluenza 2 was low and the number of positive specimens for these viruses was too small. Nucleic acids were extracted by using a QIAamp Viral RNA mini kit (Qiagen, Hilden, Germany) and a QIAcube platform (Qiagen).
This study was conducted under the approval of the Institutional Review Board of Dongtan Sacred Heart Hospital (2015-069), Hwaseong, Korea.

Gene expression of CD137 and CD137L in human colon cancer tissue and PBMCs was determined using quantitative real-time PCR (RT-qPCR). Total cellular RNA was extracted using RNeasy Minikit (Qiagen, Hilden, Germany) on the QIAcube platform (Qiagen) according to the manufacturer’s instructions. Reverse transcription was performed using ImProm II reverse transcriptase system (Promega, Mannheim, Germany) and Eppendorf Mastercycler (Eppendorf, Hamburg, Germany). For analysis of colon tissue samples, human colon-matched cDNA was purchased from Pharmingen (Heidelberg, Germany) and used as a control. For analysis of patients’ PBMCs, samples from eight healthy probands were used as a control. PCR was performed using Taqman Gene Expression Master Mix (Life Technologies, Carlsbad, CA) and Taqman Gene Expression Assay (Life Technologies) in concordance to the manufacturer´s instructions and carried out in duplicates on a Biorad CFX96 Touch Real-Time PCR Detection System. Housekeeping genes β-actin, B2M (β 2 microglobulin), and RPLP0 (ribosomal protein, large, P0) were used for relative quantification. Reproducibility was confirmed by three independent PCR runs. The relative gene expression value, fold difference (FD), is expressed as 2^−ΔΔCq.