Copy Number Variation Sequencing for Comprehensive Diagnosis of Chromosome Disease Syndromes

Abstract

Detection of chromosome copy number variation (CNV) plays an important role in the diagnosis of patients with unexplained clinical symptoms and for the identification of chromosome disease syndromes in the established fetus. In current clinical practice, karyotyping, in conjunction with array-based methods, is the gold standard for detection of CNV. To increase accessibility and reduce patient costs for diagnostic CNV tests, we speculated that next-generation sequencing methods could provide a similar degree of sensitivity and specificity as commercial arrays. CNV in patient samples was assessed on a medium-density single nucleotide polymorphism array and by low-coverage massively parallel CNV sequencing (CNV-seq), with mate pair sequencing used to confirm selected CNV deletion breakpoints. A total of 10 ng of input DNA was sufficient for accurate CNV-seq diagnosis, although 50 ng was optimal. Validation studies of samples with small CNVs showed that CNV-seq was specific and reproducible, suggesting that CNV-seq may have a potential genome resolution of approximately 0.1 Mb. In a blinded study of 72 samples with known gross and submicroscopic CNVs originally detected by single nucleotide polymorphism array, there was high diagnostic concordance with CNV-seq. We conclude that CNV-seq is a viable alternative to arrays for the diagnosis of chromosome disease syndromes.

Maternal X chromosome copy number variations are associated with discordant fetal sex chromosome aneuploidies detected by noninvasive prenatal testing

“…To investigate a possible genetic cause of these two discordant NIPT results, we further analyzed the maternal WBCs stored from the original NIPT blood samples by CNV-Seq, which we have previously shown to be reliable, accurate, quantitative and rapid for detecting low levels of X chromosome mosaicism [12] and small CNVs associated with chromosome disease syndromes [15]. In the first sample SCA001, CNV-Seq detected four copies of a 1.69 Mb Xp22.31 segment (Fig.…”

“…CNV maps can be derived using high-density oligonucleotide or SNP arrays which are designed specifically for pathogenic CNV analysis [19]. However, CNVSeq which was used in this study may be more suitable for this purpose because the method not only displays a high chromosome resolution of 0.1 Mb, but can also accurately quantitate the CNV [15] allowing precise adjustment of fetal NIPT CNV z-scores and thus a more accurate diagnosis of fetal CNVs. Therefore, for future CNV testing by NIPT, it should be possible, as for SCAs, to also reduce the number of patients referred for invasive testing when the fetus has been identified with a potentially pathogenic CNV.…”

“…Copy number variation sequencing with a resolution of approximately 0.1 Mb for genomic DNA [12,15] was used to determine the maternal karyotype. White blood cells (WBCs) frozen from the original NIPT blood samples were used as the source of maternal DNA for sequencing.…”

Characterization of chromosomal abnormalities in pregnancy losses reveals critical genes and loci for human early development

“…DNA was extracted from tissues using DNeasy Blood & Tissue Kit (#69506; Qiagen, Germantown, MD) and purified with Genomic DNA Clean & Concentrator Kit (#D4011; Zymo, Research, Irvine, CA). The sequencing and calling of CNVs were performed as previously described (Liang et al., ; Liu et al., ; Wang et al., ). Briefly, about three million sequencing reads of each sample were mapped to the reference genome using the Burrows‐Wheeler Aligner (BWA) and allocated to 20 kb sequencing bins with a 5‐kb sliding to achieve a higher resolution in identifying CNVs.…”

Clinical application of next-generation sequencing in preimplantation genetic diagnosis cycles for Robertsonian and reciprocal translocations

“…The quality control criteria for judging successful WGA included a concentration of >40 ng/μl (20 μl final volume after column purification) and a size range of 100-500 bp. A total of 50 ng of WGA product was used as a template for CNV-Seq [24,25]. WGA products were fragmented to an average size of 300 bp and end-ligated with 9-bp barcoded sequencing adaptors [26].…”