Poster abstracts
Poster number 40 submitted by Cameron Divoky
mmOligo: Direct Sequencing of Small RNAs via LC-MS/MS
Cameron Divoky (OSU, Biological Chemistry and Pharmacology ), Olivia Miller (OSU, Biological Chemistry and Pharmacology ), Mike Freitas (OSU, Biological Chemistry and Pharmacology ), Herman Singh, Mike Kolich, John Rose, Andrii Albatov, Nickolai But (Mass Matrix)
Abstract:
Next-generation sequencing (NGS) of RNA can generate large amounts of sequencing data, however, current NGS methods often mask post-transcriptional modifications (PTMs) or limit the scope of modifications that can be identified on a given RNA. While LC-MS/MS has been used to identify a broader range of RNA modifications, this is typically achieved through enzymatic digestion of the RNA, limiting the analysis to only include the nucleosides and thereby removing the sequence context in which the modifications occur. mmOligo, a bioinformatic tool, was developed to bridge this gap and enable the direct sequencing of RNAs via LC-MS/MS while preserving the sequence context in which modifications occur. Using a commercially available small RNA isolation kit and OSU’s Mass Spectrometry Facility, we have established a pipeline for extracting Argonaute-associated small RNAs (smRNAs) from mammalian cells and analyzing these smRNA pools via LC-MS/MS and mmOligo. Preliminary LC-MS/MS data indicate that complex RNA pools can be reliably separated by LC, and a mass analyzer optimized for negative-ion mode can generate sufficient sequencing data for mmOligo analysis. For this symposium, our mmOligo software was benchmarked against two currently available direct-RNA sequencing algorithms for LC-MS/MS data—Pytheas and the Nucleic Acid Search Engine (NASE)—to demonstrate mmOligo’s improved sequencing speed with large datasets, its ability to handle a greater number of variable modifications, and its capability to distinguish between sugar and base modifications that result in identical mass shifts of the parent ions.
Keywords: direct-sequencing, RNA-modifications, small RNAs