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[Preprint]. 2024 May 28:2023.04.06.535889.
doi: 10.1101/2023.04.06.535889.

Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis

Caroline A McCormick  1 , Stuart Akeson  1 , Sepideh Tavakoli  1 , Dylan Bloch  1 , Isabel N Klink  1 , Miten Jain  1   2 , Sara H Rouhanifard  1
Affiliations

Multicellular, IVT-derived, unmodified human transcriptome for nanopore-direct RNA analysis

Caroline A McCormick et al. bioRxiv. .

Update in

Abstract

Nanopore direct RNA sequencing (DRS) enables measurements of RNA modifications. Modification-free transcripts are a practical and targeted control for DRS, providing a baseline measurement for canonical nucleotides within a matched and biologically derived sequence context. However, these controls can be challenging to generate and carry nanopore-specific nuances that can impact analysis. We produced DRS datasets using modification-free transcripts from in vitro transcription (IVT) of cDNA from six immortalized human cell lines. We characterized variation across cell lines and demonstrated how these may be interpreted. These data will serve as a versatile control and resource to the community for RNA modification analysis of human transcripts.

Keywords: Bioinformatics; Transcriptomics.

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Conflict of interest statement

COMPETING INTEREST STATEMENT The authors declare no competing interests.

Figures

Figure 1.
Figure 1.
Alignment performance and coverage comparison. (A) Average percent identity of aligned DRS reads to Gencode.v38 Transcript sequences against read length (nts); data generated with Nanoplot[21]. (B) Percent of observed protein-coding transcripts (out of total protein-coding transcripts) against transcript minimum read count cut-off for each cell line. pan-IVT is the additive combination of all cell line coverage (C) Target cell line gene representation as a function of reads sampled from five sample population cell lines. Each cell line was used as the target cell line; the line on the graph corresponds to the representation of the target cell line listed in the legend.
Figure 2.
Figure 2.
Recommended Analysis Inclusion Criteria (A) Decision tree to determine if a position should be considered for downstream analysis. (B) Sanger sequencing for orthogonal support determines suitability for downstream modification analysis. Comparing HeLa biological RNA (DRS), IVT RNA (DRS), gDNA (Sanger sequencing), and genome reference (GRCh38). Red bars indicate exclusion from downstream analysis, and green bars indicate inclusion.
Figure 3
Figure 3
(A). Ionic Current distributions for MCM5 (chr22:35424407 +/− 4) across all 6 cell lines.
See this image and copyright information in PMC

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