A novel quantitative targeted analysis of X-chromosome inactivation (XCI) using nanopore sequencing

Abstract

X-chromosome inactivation (XCI) analyses often assist in diagnostics of X-linked traits, however accurate assessment remains challenging with current methods. We developed a novel strategy using amplification-free Cas9 enrichment and Oxford nanopore technologies sequencing called XCI-ONT, to investigate and rigorously quantify XCI in human androgen receptor gene (AR) and human X-linked retinitis pigmentosa 2 gene (RP2). XCI-ONT measures methylation over 116 CpGs in AR and 58 CpGs in RP2, and separate parental X-chromosomes without PCR bias. We show the usefulness of the XCI-ONT strategy over the PCR-based golden standard XCI technique that only investigates one or two CpGs per gene. The results highlight the limitations of using the golden standard technique when the XCI pattern is partially skewed and the advantages of XCI-ONT to rigorously quantify XCI. This study provides a universal XCI-method on DNA, which is highly valuable in clinical and research framework of X-linked traits.

Incorporating Nanopore Sequencing into a Diverse Diagnostic Toolkit for Incontinentia Pigmenti

“…Traditional analysis of fragment lengths after methylation sensitive restriction enzyme digest at the AR and RP2 loci is frequently employed for analysis of skewed XCI. Most recently, this has also been achieved using ONT sequencing after CRISPR-Cas9 enrichment, demonstrating a series of advantages towards traditional diagnostic strategies and reliably and reproducibly determining the XCI status in multiple females 30 . Since ONT sequencing relies on long-reads and allows for pinpointing a variant’s location within a gene, even in the presence of closely related pseudogenes, it can facilitate both variant mapping and XCI analysis, even extending to the precise assignment of the variant to the predominantly active or inactive X-chromosome.…”

The Next, Next-Generation of Sequencing, Promising to Boost Research and Clinical Practice

“…This allows for parallel interrogation of the epigenome and considerations such as genetic imprinting 56 and X-chromosome inactivation. 57 Using an ultra-rapid Nanopore sequencing protocol and methylation profiling, childhood central nervous system tumors were classified within 40 minutes, to assist with intraoperative neurosurgical decision-making. 58 As long-read quality, capability, and throughput are set to keep increasing, providing greater confidence in calling variants that short-read sequencing can and cannot resolve, this modality has the potential to supplant most other currently available clinical genetic tests in one high-confidence test.…”

Nanopore sequencing-based episignature detection

“…Methylation was visualized with Methylartist [21] and Integrative Genomics Viewer (IGV) [22]. To evaluate XCI, we assessed methylation at two validated loci, also recently targeted by CRISPR-Cas9 nanopore sequencing for this purpose: 115 CpGs at the AR gene loci (chrX:67543761-67546170, hg38) and 57 CpGs at the RP2 gene loci (chrX:46836539-46837273, hg38) [23]. In addition, 99 CpGs in the promotor region of PHF6 were evaluated (chrX:134372110-134374891, hg38).…”

“…Moreover, the generated long reads enable haplotyping [12], allowing the phasing of genetic and epigenetic variation. Recent studies showed the ability of nanopore sequencing to detect X-chromosome inactivation (XCI) [13] as well as methylation disturbances at imprinted loci [14] and short tandem repeats causing DD [15].…”