Is It Possible To Detect All Segmental Chromosomal Imbalances With Ngs Using Custom Analysis Algorithms?

Next Generation Sequencing (NGS) has been used for PGT of chromosome rearrangements with a resolution of ≥ 5-20 Mb, which is declared detection limit of available commercial kits. However, many patients carry a chromosome rearrangement below these detection limit values thus a customized analysis approach enables effective use of this method for a broad range of chromosomal imbalances. In this study we aimed to detect small unbalanced chromosome segments (≥1 MB) below manufacturer’s recommended resolution limits. NGS library of genomic DNA from 4 patients with unbalanced segmental imbalances were prepared using PG-Seq Kit 2.0 (PerkinElmer), 48 samples were sequenced with MiSeq (Illumina) and analysed with PG-Find Analysis Software. Six small unbalanced chromosome segments previously detected with Microarray were evaluated with the sizes of 0.5 MB to 4.5 MB. Three of patients included in the study had products of familial subtelomeric translocations and one of them had a product of familial paracentric inversion. Reanalysis was performed for samples, where standard analysis fails to detect target rearrangements with specific analysis algorithm to focus on target regions by altering filtering paramateres. Each sample was assessed for the size of the segmental gain/loss (Mb), copy number for each segment, concordance with Microarray results and was compared in terms of different algorithms we used. NGS was capable of detecting 7/8 (87.5%) unbalanced segments previously identified using Microarray. 6/8 (75%) of variants were ≤4.5 Mb. The rest of variants were 8.6 MB and 17.9 MB respectively (Table 1). One of the variants was very small (0.5MB) that we couldn’t detect by NGS. According to assessment of individual data quality, optimum number of reads was 500000 per sample to detect segmental gains/losses ≥1 MB. This study has shown that analysis with different filtering algorithms makes NGS qualified to detect unbalanced chromosome rearrangements ≥1 Mb. Further studies can be designed with lower sample size (24) in order to increase read/sample, which may enable detection of chromosomal imbalances bellow 1 MB. Further studies with PGT samples should be required to define the detection limit of NGS method.Table 1True positive detection of CNV’s with customized analysis algorithm is presented.CNVSizeStandard AlgorithmCustomized Algorithm1,4 MB17,9 MBNOYESYESYES2,6 MB1,15 MBNONOYESYES1,2 MB4,5 MBNONOYESYES8,6 MB0,5 MBYESNOYESNO Open table in a new tab