ABSTRACT
The novel coronavirus disease 2019 (COVID-19) pandemic poses a serious public health risk. In this report, we present a modified sequencing workflow using short tiling (280bp) amplicons library preparation method paired with Illumina's iSeq100 desktop sequencer. We demonstrated the utility of our workflow in identifying gapped reads that capture characteristics of subgenomic RNA junctions within our patient cohort. These analytical and library preparation approaches allow a versatile, small footprint and decentralized deployment that can facilitate comprehensive genetics characterizations during outbreaks. Based on the sequencing data, Taqman assays were designed to accurately capture the quantity of subgenomic ORF5 and ORF7a RNA from patient samples and demonstrated utility in tracking subgenomic titres in patient samples when combined with a standard COVID-19 qRT-PCR assay.
ABSTRACT
BACKGROUND: The standard of care for thyroid cancer management is thyroidectomy and adjuvant radioactive iodine (RAI). There is a paucity of clinical tool that quantifies residual thyroid volume reliably for precise adjuvant RAI dosing. Serum thyroglobulin (TG), tumour marker for thyroid cancer, takes 4 weeks for complete clearance due to its long half-life, and might be undetectable in 12% of structural disease patients. It detects recurrence with a sensitivity of 19-40%, mainly attributed to issue of TG antibody interference with TG immunometric assay. We hypothesise that the quantity of thyroid-specific cell-free RNA (cfRNA) is indicative of amount of thyroid tissues, and that during thyroid surgery, cfRNA levels decrease accordingly. METHODS: We identified 11 biologically significant and highly expressed thyroid-specific targets from Human Protein Atlas and literature. To assess for a fall in thyroid-specific cfRNA level, we recruited 16 patients undergoing thyroid surgery or RAI for malignant or benign thyroid disease, and tracked longitudinal trend of cfRNA. To assess the utility of cfRNA in detecting metastatic thyroid cancer, cfRNA of 11 patients at intermediate to high risk of recurrence was measured during surveillance and at time of clinical recurrence. RESULTS: The multiplex assay was capable of amplifying and quantifying multiple thyroid-specific genes in a single reaction. The selected targets were amplified successfully from RNA extracted directly from the thyroid (positive control), indicating that they were highly expressed within thyroid tissue. These cfRNAs were present in plasma, in amounts quantifiable using qRT-PCR. Four cfRNA transcripts (TPO, GFRA2, IVD, TG) fell post-treatment in more than 50% of cohort. The thyroid peroxidase (TPO) cfRNA fell post-therapy in 63% of cohort by 80%, as early as 1 day post-treatment, supporting the potential role as early indicator of remnant thyroid tissue volume. We demonstrated the clinical relevance of circulating TPO cfRNA by tracking temporal changes in setting of peri-treatment, recurrence, and TG Ab positive state. CONCLUSION: Using a multiplex pre-amplification approach, the TPO cfRNA was a potential biomarker that can track residual thyroid mass. It can be further optimised for quantification of thyroid volume to guide RAI doses and for detection of thyroid cancer recurrence.
