![]() For false positive isoforms with a known count of 0 TPM distribution is plotted and the % of TPMs mapped to 0 count isoforms shown. TPM coefficient of variation shown for isoforms with a known concentration of 1 fmol. ( C) SIRV Mix E0 Over annotation (O) isoforms. ( B) SIRV Mix E2 Complete annotation (C) isoforms (Spearman's r correlation shown). ( A) SIRV Mix E0 Complete annotation (C) isoforms (coefficient of variation (CV) of TPMs shown). SIRV isoform expression quantified as TPM(log e), i.e.: log e(TPM + 1). (A–D) Comparison of NanoCount, Salmon and StringTie2 for quantification of SIRV spike-in isoform mixes. X-axis truncated at 0.5.Ĭomparison of methods for quantifying DRS spike-in controls. Dotted line represents 95% cutoff for full-length reads. Fraction of known transcript length covered by each read. ![]() ( F) Fraction of full-length SH-SY5Y reads. Trend line was plotted using a generalized additive model, an extension of a generalised linear model where the linear form is replaced by sum of smooth functions. ( E) Fraction of known transcript length covered by each read (coverage fraction) compared to known transcript length. D2T1 & T2-technical replicates of differentiated replicate sample 2. ![]() U1 & U2-undifferentiated replicates 1 & 2. Lower coverage at extreme 3' corresponds to soft clipping of the first bases sequenced which often have lower phred quality. Lines show mean coverage across all genes across the length of the gene body. Length of all genes normalised to 100 and plotted from 5′ (0) to 3′ (100). ( D) Gene body coverage of SH-SY5Y reads in each sample. ( C) Length of all SH-SY5Y and sequin pass reads. The primary alignment (P) is now the remaining alignment with the highest AS. In this example the read count is split 0.7 to 0.3 between the two remaining alignments. Finally, the expectation-maximisation algorithm is initiated to quantify isoforms. Next, alignments with an alignment score (AS) <95% of the highest remaining AS are discarded. ![]() Alignments with a 3′ end >50nt from the read 3′ end are discarded (grey). A nanopore read (black) and example alignments (blue) are shown. Reads were analysed to identify and quantify genes and transcript isoforms and their differential expression. ![]() Native polyA purified SH-SY5Y RNA was combined with ‘sequin’ spike-in RNA, prepared for DRS and sequenced on an Oxford Nanopore MinION. Cultured SH-SY5Y cells were differentiated in triplicate and RNA extracted from undifferentiated and differentiated cells. Published by Oxford University Press on behalf of Nucleic Acids Research.Įxperimental overview and DRS read metrics. Our results demonstrate enhanced DRS isoform quantification with NanoCount and establish the ability of DRS to identify biologically relevant differential expression of genes and isoforms. NanoCount quantification of thousands of novel isoforms discovered with DRS likewise enabled identification of their differential expression. Genes upregulated in neuron-like cells were associated with neurogenesis. Differential expression of 231 genes, 333 isoforms, plus 27 isoform switches were detected between undifferentiated and differentiated SH-SY5Y cells and samples clustered by differentiation state at the gene and isoform level. Using synthetic controls and human SH-SY5Y cell differentiation into neuron-like cells, we show that DRS accurately quantifies RNA expression and identifies differential expression of genes and isoforms. We developed NanoCount for fast, accurate transcript isoform quantification in DRS and demonstrate it outperforms similar methods. However, there are a lack of tools specifically designed for DRS and its ability to identify differential expression in complex organisms is poorly characterised. Sequencing full-length native RNAs using long-read direct RNA sequencing (DRS) has the potential to overcome many limitations of short and long-read sequencing methods that require RNA fragmentation, cDNA synthesis or PCR. Accurately quantifying gene and isoform expression changes is essential to understanding cell functions, differentiation and disease. ![]()
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