4. VALIDATION STUDY OF A NGS-BASED PGT-A WORKFLOW FOR MOSAICISM AND SEGMENTAL COPY NUMBER VARIATION

摘要: Introduction Recently, with the introduction of Next Generation Sequencing (NGS), preimplantation genetics test for aneuploidy (PGT-A) has been greatly advanced. While copy number variation (CNV) calling full chromosome aberrations well developed by many NGS-based PGT-A workflows, algorithms mosaicism and segmental CNVs are still not established. In this study, we assess specificity accuracy a newly developing workflow CNV. Material & methods Thirteen Coriell cell lines known karyotypes were analyzed in study. To mosaic calling, 5 cells precisely picked under dissection microscope processed using protocol Ion ReproSeq™ PGS Kits GeneStudio S5™ System (Thermo Fisher Scientific). Mosaic samples 0%, 20%, 40%, 60%, 80% 100% generated combining defined from two lines. For evaluating CNV 4-5 each sampled. The sequence data was decimal ploidy value (mosaic) algorithm Reporter v5.10. This combined either default (2Mb), 1Mb or 0.5Mb tile size reference baseline. total, 144 4 replications sample 3 all other samples. Results evaluate sensitivity sets mixing (i) 46,XY 47,XX,+21, (ii) 46,XY,del(4p) 47,XY,+13 profiles Chr13 closely matched expected values at tested combinations, contrast, those Chr21 showed 60% but 20% 40% groups. Meanwhile, baseline could detect loss del(4p)27Mb, displayed 9%, 15% 17 % overestimation groups, respectively. By choosing different baselines while keeping parameters values, ability to With baseline, trisomy Chr13, Chr18, ChrX, monosomy Chr21, dup(3q)99.1Mb, del(5p)32Mb, del(4p)27Mb dup(6p)20.97Mb be correctly called. dup(22q)14.75Mb dup(9p)11Mb dup(16p) 3.6Mb, del(5q) 2.3Mb, dup(6p) 2.19Mb, del(9p) 1.81Mb del(14q) 1.18 Mb, consistently reported breakpoints. Of note, identified some unexpected events various sizes locations, such as dup(1q)4.6Mb, del(15q)2.6Mb, del(4p)1.4Mb, del(17q)1.4Mb, dup(2q)0.9Mb, del(22q)0.8Mb del(15q)0.7Mb. Conclusion Accuracy both callings may vary among chromosomes, regions chromosomes. Segmental is less sensitive accurate than calling. Choosing higher resolution enhance small reduce specificity. Further modification optimization needed. study provides useful information establishing guidelines.