COVID-19

Whole Exome Sequencing (WES)

Genomic DNA is first evaluated for its quantity, and quality, using Agilent TapeStation 4200. A DIN (DNA integrity number) of 8 or higher is expected. One hundred to two hundred nanograms of gDNA are used for library preparation. The library preparation is performed using Biomek FXP automation for up to 96 specimens per plate (Beckman Coulter). Briefly, DNA library preparation includes DNA fragmentation with Covaris ME220 AFA sonicator to roughly 300bp, end-repair, dA tailing, ligation of index adaptors, and amplification, following the SureSelect XTHS Target Enrichment System for Illumina Paired-End Multiplexed Sequencing Version A1, July 2017 (Agilent Technologies). Libraries are then hybridized, captured, and amplified with Agilent Human All Exon V7 probe set (48Mb, hg38) manually. Each resulting captured library is quantified and its quality accessed by Qubit and Agilent Bioanalyzer. Libraries are then pooled in equal molarity. Average size of library insert is about 250-400bp. The pooled libraries are then denatured, and neutralized, before loading on to NovaSeq 6000 sequencer at 300pM final concentration for 150bp paired-end sequencing (Illumina, Inc.). Approximately 35M reads per library will be generated for about 100-fold coverage. A Phred quality score (Q score) is used to measure the quality of sequencing. More than 95% of the sequencing reads reach Q30 (99.9% base call accuracy).

RNA Transcriptomics

Total RNA is first evaluated for its quantity, and quality, using Agilent Bioanalyzer 2100. For RNA quality, a RIN number of 7 or higher is desired. One hundred nanograms of total RNA are used. The library preparation is performed using Biomek FXP automation for up to 96 specimens per plate (Beckman Coulter). Briefly, cDNA library preparation includes PolyA-RNA purification/enrichment, RNA fragmentation, cDNA synthesis, ligation of index adaptors, and amplification, following the KAPA mRNA Hyper Prep Kit Technical Data Sheet, KR1352 – v4.17 (Roche Corporate). Each resulting indexed library with average insert size of 250-400bp is quantified and its quality accessed by Agilent TapeStation, and multiple libraries are pooled in equal molarity with QIAgility (Qiagen). The pooled libraries are then denatured, and neutralized, before loading on to NovaSeq 6000 sequencer at 300pM final concentration for 150bp paired-end sequencing (Illumina, Inc.). Approximately 30-40M reads per library are generated. A Phred quality score (Q score) is used to measure the quality of sequencing. More than 95% of the sequencing reads reach Q30 (99.9% base call accuracy).

Mapping QC and data analysis:

The sequencing data are assessed using FastQC (Babraham Bioinformatics, Cambridge, UK) for quality control.

Chemokine/cytokine multiplex analyses

Plasma specimens were aliquoted and frozen at -80C for multiplex batch analyses by the IUSCCC/CTSI MultiPlex Analysis Core (MAC). The MAC uses Luminex xMAP technology and the Bio-Plex 200 System with High Throughput Fluidics (HTF) Multiplex Array System (Bio-Rad Laboratories, Hercules, CA) with Bio-PlexTM 6.0 Manager software (Bio-Rad) to analyze specimens. specimens were thawed on ice, centrifuged to remove particulates (10,000 x g for 10 min), diluted as appropriate and assayed in duplicate for simultaneous quantification of 48 cytokines/chemokines using the MILLIPLEXHCYTA-60K-PX48 Human Cytokine/Chemokine/Growth Factor Panel A 48 Plex Kit (Millipore, Billerica, MA), which detects the following cytokines:

sCD40L, EGF, Eotaxin, FGF-2, FLT-3L, Fractalkine, G-CSF, GM-CSF, GROα, IFNα2, IFNγ, IL-1α, IL-1β, IL-1RA, IL-2, IL-3, IL-4, IL-5, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12 (p40), IL-12 (p70), IL-13, IL-15, IL-17A, IL-17E/IL-25, IL-17F, IL-18, IL-22, IL-27, IP-10, MCP-1, MCP-3, M-CSF, MDC, MIG, MIP-1α, MIP-1β, PDGF-AA, PDGF-AB/BB, RANTES, TGFα, TNFα, TNFβ, VEGF-A

PBMC Analyses

Peripheral blood mononuclear cells (PBMCs) from the COVID patients were subjected to cell surface staining and intracellular staining (ICS) to determine immune cell phenotype, activation, and immune response. For cell surface staining, frozen PBMCs were thawed and then incubated with fixable viability dye (eBioScience, ThermoFisher Scientific) to discriminate live and dead cells. To characterize different immune cell populations, PBMCs were further stained with fluorochrome-conjugated antibodies against cell lineage markers (CD3, CD4, CD11c, CD14, CD16, CD56, and CD123, CD127, and the human MR1 tetramers loaded with a potent MAIT cell ligand 5-OP-RU (5-(2-oxopropylideneamino)-6-D-ribitylaminouracil) and cell activation markers (CD25, CD38, CD56, CD69, and HLA-DR). To analyze B cell subsets, PBMCs were stained with fixable viability dye and fluorochrome-conjugated antibodies against B cell markers (CD19, CD20, CD24, CD27, CD38, CD138, and IgD) and activation markers (BTLA, CD25, CD69, CD86, FcRL4, PD-1, and HLA-DR). Stained cells were fixed with 2% paraformaldehyde (PFA) before flow analysis. For ICS, PBMCs were stimulated for 5.5 hours with the pharmacological agents phorbol 13-acetate 12-myristate (PMA) and ionomycin at a concentration of 100 ng/ml and 1 M, respectively, together with the protein transport inhibitor GoljiStop (BD Biosciences, San Jose, CA). Stimulated cells were stained with fixable viability dye and fluorochrome-conjugated antibodies against cell lineage markers (CD3, CD4, CD8, CD56, CD161, and the MR1 tetramer loaded with 5-OP-RU). Stained cells were then fixed were and permeabilized with the Cytofix/Cytoperm reagents (BD Biosciences, San Jose, CA), followed by staining with antibodies against cytokines (IFN- TNF-a, IL-10, and IL-17), and the cytotoxic molecule granzyme B. Appropriate isotype controls were used at the same protein concentration as the test antibodies for control staining. All antibodies were purchased from BioLegend, San Diego, CA), and the human MR1 tetramers were produced by the NIH Tetramer Core Facility as permitted to be distributed by the University of Melbourne. Stained cells were subsequently acquired using a BD LSRFortessa flow cytometer (BD Biosciences, San Jose, CA). Flow data were analyzed using FlowJo v10 software (Tree Star, San Carlos, CA).