Exploiting nanopore sequencing for characterization and grading of IDH‐mutant gliomas

Abstract

The 2021 WHO Classification of Central Nervous System Tumors recommended evaluation of cyclin‐dependent kinase inhibitor 2A/B (CDKN2A/B) deletion in addition to codeletion of 1p/19q to characterize IDH‐mutant gliomas. Here, we demonstrated the use of a nanopore‐based copy‐number variation sequencing (nCNV‐seq) approach to simultaneously identify deletions of CDKN2A/B and 1p/19q. The nCNV‐seq approach was initially evaluated on three distinct glioma cell lines and then applied to 19 IDH‐mutant gliomas (8 astrocytomas and 11 oligodendrogliomas) from patients. The whole‐arm 1p/19q codeletion was detected in all oligodendrogliomas with high concordance among nCNV‐seq, FISH, DNA methylation profiling, and whole‐genome sequencing. For the CDKN2A/B deletion, nCNV‐seq detected the loss in both astrocytoma and oligodendroglioma, with strong correlation with the CNV profiles derived from whole‐genome sequencing (Pearson correlation (r) = 0.95, P < 2.2 × 10−16 to r = 0.99, P < 2.2 × 10−16) and methylome profiling. Furthermore, nCNV‐seq can differentiate between homozygous and hemizygous deletions of CDKN2A/B. Taken together, nCNV‐seq holds promise as a new, alternative approach for a rapid and simultaneous detection of the molecular signatures of IDH‐mutant gliomas without capital expenditure for a sequencer.

iSCORED: nanopore-based random genomic sampling for intraoperative molecular diagnosis

“…2b). Furthermore, the output is approximately threefold the data volume of the recently published SMURF-based method (nCNV-seq) within the same 50-60 minutes sequencing window 22 . Across the 26 investigated samples, the diagnostic accuracy of iSCORED platform was 100% in detecting gene amplification of more than 10 copies (95% confidence interval 31 : 91%-100%, Pearson r = 0.81 by comparing to the NGS results; Table 1).…”

“…1f, Additional table 1). While the SMURF approach demonstrates a slightly better reconstruction efficiency (14.5), its sequential approach requires a substantial input DNA (2-3 μg) 21 and an extended preparation time (90-120 minutes) 21,22 . This in contrast to the iSCORED method, which requires only 200-400 ng of input gDNA and a preparation time of 30 minutes.…”

“…By sequencing a fraction of randomly assembled genomic fragments, the genome-wide chromosomal integrity can be quantitatively assessed. While previous attempts using long concatenated reads for quantitative genomic analysis have been introduced, the methods are lengthy and require sequential mechanical shearing (SMASH 20 ) or enzymatic digestion (SMURF 21,22 ) followed by DNA purification, and ligation. The multi-step preparation requires high amount of input DNA and takes a few hours for processing.…”

Cellular Components of the Tumor Environment in Gliomas—What Do We Know Today?

“…A comparatively new sequencing technique is nanopore sequencing which does not require a preceding polymerase chain reaction or labeling of the sample. Nanopore sequencing has occasionally been used to study gliomas, one example being the detection of mutations of the gene for the "cyclin-dependent kinase inhibitor 2A/B (CDKN2A/B) in IDH-mutated gliomas [144]. This method has yet to be applied to TEM cells.…”

“…This method has yet to be applied to TEM cells. A challenge is still the comparatively high error rate in the reading of the base sequence, but this is constantly being improved by increasing the reading length of the sequences [144]. The outstanding new method for examining the cells of the TEM is the complex of single-cell analysis, which can basically be carried out in three different ways.…”