Primers flanking specific fusions were designed based on the EML4‐ALK fusion breakpoint sequences identified (EML4ex13_F‐GTGCAGTGTTTAGCATTCTTG and ALKex 20_R–GTAGTCGGTCATGATGGTCG) and used to perform reverse transcription using the I Script cDNA Synthesis Kit (Bio‐Rad, Hercules, California) and 500 ng of RNA from the FNA specimen. Total RNA (50‐250 ng) was reverse transcribed to cDNA. 2014;10:e1003665. The quality of DNA was assessed using the Qubit 2.0 fluorimeter and the Qubit dsDNA HS Assay Kit. Hum Mol Genet. A representative ALK FISH‐positive case (L‐3) that had a sufficient amount of RNA also was confirmed by RT‐PCR followed by Sanger sequencing. endobj 2538 0 obj While SNV detection from abundant single-cell RNA sequencing (scRNA-seq) data is applicable and cost-effective in identifying expressed variants, inferring sub-clones, and deciphering genotype-phenotype linkages, there is a lack of computational methods specifically developed for SNV calling in scRNA-seq. ex indicates exon; Neg Contr, negative control; NTC, no template control. The data from both were analyzed using the Ion Torrent Suite Software followed by a laboratory‐developed pipeline. [278 0 0 0 0 0 0 0 0 0 0 0 0 333 278 278 556 556 556 556 556 556 556 556 556 556 0 0 0 0 0 0 0 667 667 722 722 667 611 778 722 278 500 0 556 833 722 778 667 0 722 667 611 0 667 0 0 0 0 0 0 0 0 556 0 556 556 500 556 556 278 556 556 222 0 0 222 833 556 556 556 0 333 500 278 556 500 722 500 500] <>/Font<>/ProcSet[/PDF/Text]/XObject<>>>/Rotate 0/Type/Page>> Areas with the highest percentage of tumor cells and least amount of contaminating materials (nonneoplastic cells, debris, and mucin) were selected for testing. endobj (Bottom) DNA‐based NGS assay. Universal patterns of selection in cancer and somatic tissues. endobj <> <> Note. [5722 0 R 5723 0 R] Figure 3 demonstrates 2 representative cases of PTC with the same fusion (CCDC6‐RET) and mutation (BRAF p.V600E) detected in the FNA specimen and paired FFPE tissue. BMC Syst Biol. All told, the researchers observed more than 31 million confirmed SNVs. In conclusion, cytology specimens are a reliable resource for RNA‐based molecular testing and can be used to detect multiple types of genomic aberrations simultaneously from limited cytology materials to aid in the management of patients with advanced cancer. 2554 0 obj endobj There was 100% concordance between the FISH and RNA findings. Using the same assay, common SNVs and small deletions were detected in BRAF, KRAS, and EGFR, including coexisting tyrosine kinase inhibitor (TKI)‐sensitizing and TKI‐resistant mutations (p.L858R and p.T790M in case L‐9). It is important to note that the frequency of variants in the current study cohort does not represent that in the general tumor population because cases with known aberrations were preferentially selected for the purposes of test validation. Du Y, Thompson EG, Muller J, Valvo J, Braun J, Shankar S, van den Berg RA, Jongert E, Dover D, Sadoff J, Hendriks J, Gardner MJ, Ballou WR, Regules JA, van der Most R, Aderem A, Ockenhouse CF, Hill AV, Wille-Reece U, Zak DE. Learn about our remote access options, Department of Pathology, University of Iowa Hospitals and Clinics, Iowa City, Iowa, Department of Pathology, King Hussein Cancer Center, Amman, Jordan.
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