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Updated: May 22, 2026
Targeted Next-generation Sequencing and Bioinformatics Pipeline to Evaluate Genetic Determinants of Constitutional Disease
Published on: April 4, 2018
Determination of RET Sequence Variation in an MEN2 Unaffected Cohort Using Multiple-Sample Pooling and
R L Margraf1, J D Durtschi, J E Stephens
1Research & Development, ARUP Institute for Clinical and Experimental Pathology, 500 Chipeta Way, Salt Lake City, UT 84108, USA.
Abstract:
Multisample, nonindexed pooling combined with next-generation sequencing (NGS) was used to discover RET proto-oncogene sequence variation within a cohort known to be unaffected by multiple endocrine neoplasia type 2 (MEN2). DNA samples (113 Caucasians, 23 persons of other ethnicities) were amplified for RET intron 9 to intron 16 and then divided into 5 pools of <30 samples each before library prep and NGS. Two controls were included in this study, a single sample and a pool of 50 samples that had been previously sequenced by the same NGS methods. All 59 variants previously detected in the 50-pool control were present. Of the 61 variants detected in the unaffected cohort, 20 variants were novel changes. Several variants were validated by high-resolution melting analysis and Sanger sequencing, and their allelic frequencies correlated well with those determined by NGS. The results from this unaffected cohort will be added to the RET MEN2 database.
