Updated chemistry and base-calling models improved native RNA transcriptome and modification detection for possible diagnostic use.
Evidence
This benchmarking and proof-of-concept study tested RNA004 Nanopore direct RNA sequencing with pseudouridine and N6-methyladenosine models across cell lines, synthetic oligos, human blood, and one patient with METTL5 truncating mutations.
Caveat
Clinical evidence is still limited to an initial application and benchmarking rather than validated routine diagnostic performance.
Simplified
(DRS) is a Nanopore-based technique for analyzing RNA in its native form. This technique promises breakthroughs in diagnostics and biomarker development. Coupled to RNA002 sequencing chemistry, its clinical implementation has been challenging due to low throughput, low accuracy, and lack of large-scale RNA-modification models. In this study, we evaluate the improvements achieved by pairing the latest RNA004 chemistry with novel modified-base-calling models for pseudouridine and N6-methyladenosine using diverse RNA samples from cell lines, synthetic oligos, and human blood. Finally, we present the first clinical application of DRS by confirming the loss of RNA methylation in a patient carrying truncating mutations in the METTL5. Conclusively, the combined use of RNA004 chemistry with the base-calling models significantly improved the throughput, accuracy, and site-specific detection of modifications. From this perspective, we offer an outlook on the potential suitability of DRS for use in routine diagnostics, as well as the first comprehensive benchmark of human peripheral blood. Furthermore, we demonstrate on the basis of a stop-codon readthrough enhancing agent potential roadblocks for routine quality assessments of RNA therapeutics.
Key numbers
17.3 Gb
Improvement in sequencing yield
Yield achieved by HEK293T cells using RNA004 chemistry.
1497
Detection of mA sites
Number of mA sites detected in RNA004 samples from chromosome 20.
50%
Loss of mA modification
Percentage of reads showing exon skipping in the patient with METTL5 mutations.
Full Text
We can’t show the full text here under this license.
