Analytical validation of a novel targeted next-generation sequencing assay for mutation detection in thyroid nodule aspirates and tissue

Antonella Verrienti, Valeria Pecce, Luana Abballe, Valeria Ramundo, Rosa Falcone, Farzaneh Inanloo Nigi Jak, Chiara Brunelli, Guido Fadda, Daniela Bosco, Valeria Ascoli, Raffaella Carletti, Cira Di Gioia, Giorgio Grani, Marialuisa Sponziello

Research output: Contribution to journalArticlepeer-review


Purpose: The identification of somatic mutations in cancer specimens enables detection of molecular markers for personalized treatment. We recently developed a novel molecular assay and evaluated its clinical performance as an ancillary molecular method for indeterminate thyroid nodule cytology. Herein we describe the analytical validation of the novel targeted next-generation sequencing (NGS) assay in thyroid samples from different sources. Methods: We present validation data of a novel NGS-based panel on 463 thyroid samples, including 310 fine-needle aspiration (FNA) specimens from different sources (FNA collected in preservative solution, liquid-based, and stained smears), 10 fresh frozen, and 143 formalin-fixed paraffin-embedded (FFPE) thyroid tissue specimens. Sequencing performance in the different samples was evaluated along with reproducibility, repeatability, minimum nucleic acid input to detect variants, and analytical sensitivity of the assay. Results: All thyroid samples achieved high sequencing performance, with a mean base coverage depth ranging from 2228 × (in liquid-based FNA) to 3661 × (in FNA stained smears), and coverage uniformity ranging from 86% (in FFPE) to 95% (in FNA collected in preservative solution), with all target regions covered above the minimum depth required to call a variant (500×). The minimum nucleic acid input was 1 ng. Analytic sensitivity for mutation detection was 2–5% mutant allele frequency. Conclusions: This validation study of a novel NGS-based assay for thyroid nodules demonstrated that the assay can be reliably used on multiple thyroid sample types, including FNA from different sources and FF and FFPE thyroid samples, thus providing a robust and reliable assay to genotype thyroid nodules, which may improve thyroid cancer diagnosis and care.

Original languageEnglish
Pages (from-to)451-455
Number of pages5
Issue number2
Publication statusPublished - Aug 1 2020


  • Analytical validation
  • FFPE
  • FNA
  • Next-generation sequencing
  • Thyroid cancer

ASJC Scopus subject areas

  • Endocrinology, Diabetes and Metabolism
  • Endocrinology


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