SARS-CoV-2 whole genome sequencing from pediatric clinical isolate
The retrospective cohort study enrolled pediatric patients (ages 0-20 years old) with persistent SARS-CoV-2 infection treated at Ann & Robert H. Lurie Childrens Hospital of Chicago between March 2020 and March 2023. Patients were eligible if they had 2 positive SARS-CoV-2 RT-PCR tests separated by 7 days and available residual specimens. Clinical and demographic data, including age, sex, race/ethnicity, vaccination history, comorbidities, and immunocompromising conditions, were abstracted via chart review. Immunocompromised status was defined as documented hematologic malignancy, solid or hematopoietic stem cell transplant, or active immunosuppressive therapy. Residual nasopharyngeal specimens underwent RNA extraction (QIAmp 96 Virus QIAcube HT Kit). SARS-CoV-2 viral load was quantified using the CDC RT-PCR N1 assay. Specimens with cycle threshold (Ct) <32 were subjected to whole-genome sequencing. Complementary DNA (cDNA) synthesis was performed using SuperScript IV (Thermofisher) and random hexamers. Viral genome amplification utilized the ARTIC Network V5.3.2 tiled amplicon protocol. PCR products were prepared for sequencing using the SeqWell ExpressPlex 384 kit and sequenced on an Illumina MiSeq platform (V2 500-cycle kit). Raw sequencing reads were trimmed using Trimmomatic v0.36 and aligned to the SARS-CoV-2 reference genome (MN908947.2). using BWA v0.7.18. Primer trimming was performed with iVar v1.4.3. Variant calling employed iVar with a minimum variant allele frequency (VAF) threshold of 3% and depth filter >400 reads. Consensus sequences were generated using majority base calls and deposited in GISAID. Shannon entropy was calculated per genomic position and aggregated per sample and per ORF to quantify intrahost diversity. Maximum likelihood phylogenies were constructed with IQ-TREE v2.0.5. Statistical modeling of diversity trends employed linear mixed-effects models (lme4 v1.1-35.2) adjusting for Ct value, remdesivir exposure, and time since diagnosis.