The long and the short of it: unlocking nanopore long-read RNA sequencing data with short-read differential expression analysis tools

Abstract Application of Oxford Nanopore Technologies’ long-read sequencing platform to transcriptomic analysis is increasing in popularity. However, such analysis can be challenging due to the high sequence error and small library sizes, which decreases quantification accuracy and reduces power for statistical testing. Here, we report the analysis of two nanopore RNA-seq datasets with the goal of obtaining gene- and isoform-level differential expression information. A dataset of synthetic, spliced, spike-in RNAs (‘sequins’) as well as a mouse neural stem cell dataset from samples with a null mutation of the epigenetic regulator Smchd1 was analysed using a mix of long-read specific tools for preprocessing together with established short-read RNA-seq methods for downstream analysis. We used limma-voom to perform differential gene expression analysis, and the novel FLAMES pipeline to perform isoform identification and quantification, followed by DRIMSeq and limma-diffSplice (with stageR) to perform differential transcript usage analysis. We compared results from the sequins dataset to the ground truth, and results of the mouse dataset to a previous short-read study on equivalent samples. Overall, our work shows that transcriptomic analysis of long-read nanopore data using long-read specific preprocessing methods together with short-read differential expression methods and software that are already in wide use can yield meaningful results.


INTRODUCTION
Short-read sequencing technology has underpinned transcriptomic profiling research over the past decade. The sequencing platforms offered by companies such as Illumina Inc. provide high read accuracy (>99.9%) and throughput which allows many samples to be profiled in parallel. One major limitation of short-read sequencing technology is the modest read lengths offered (currently up to 600 bases), which makes accurate isoform quantification and novel isoform discovery challenging. Long-read sequencing offers a distinct advantage in this regard, with the ability to generate reads that are typically in the 1-100 kilobase (kb) range (1), which spans the typical length distribution of spliced genes in human (for protein coding genes 1-3 kb is typical with outliers such as Titin at >80 kb) thereby allowing the sequencing of entire isoforms. This, however, comes at the expense of lower throughput and reduced accuracy compared to short-read sequencing. The two main technology platforms that dominate the field of long-read sequencing are Pacific Biosciences' (PacBio) Single-Molecule Real Time (SMRT) sequencing and Oxford Nanopore Technologies' (ONT) nanopore sequencing.
Previous work on long-read transcriptomic data focuses on transcript-level analysis, especially in the discovery of novel isoforms (2)(3)(4). Some long-read specific methods have been developed for this task. Reference-based methods, such as TALON (5), compares reads to existing gene and transcript models to create novel models. Reference-free methods, such as FLAIR (6), maps reads to the reference genome, clusters alignments into groups and collapses them into isoforms. Differential transcript usage (DTU) is another transcript-level analysis that is of great inter-est (6)(7)(8). DTU analyses examine differences in the relative proportions of expressed isoforms between two conditions. The DRIMSeq (9) method performs DTU analysis on transcript-level RNA-seq counts using a Dirichletmultinomial model. Alternatively, tools developed for differential exon usage analysis, such as the diffSplice function (10) in the limma and edgeR packages, can also be used for DTU analyses by substituting transcript-level counts for exon-level counts (11). Both DRIMSeq and diffSplice methods were developed for short-read data. The stageR package (12) can be used to control the false discovery rate (FDR) of DTU analyses through its stage-wise method which screens potential DTU genes using gene-level P-values before selecting the transcripts with evidence of DTU.
In many analyses, a gene-level analysis is as far as researchers go, so understanding how well this type of analysis can be done for long-read data is of interest. Previous studies have looked at gene-level analysis of nanopore data but study design limited the methods used. Soneson et al. (13) concluded that read coverage in native RNA libraries (∼0.5 million aligned reads per flow cell) were too low for gene-level analyses, resulting in low power and high variability. Gleeson et al. (14) found differential expression analysis performed using DESeq2 (15) was specific but not sensitive on their ONT MinION direct RNA neuroblastoma samples with synthetic spike-in controls, based on 6 samples and around 4 million reads in total. Li et al. (7) worked around this by simply using fold-changes to identify differentially expressed genes for three ONT MinION direct RNA Caenorhabditis elegans samples; however, the lack of statistical testing could lead to unreliable results. Jenjaroenpun et al. (16) used DESeq2 (15) to perform differential expression analysis on direct RNA transcript-level counts, but gene-level expression was not studied. Cruz-Garcia et al. (17) used a Snakemake pipeline which included the edgeR (18) quasi-likelihood method to identify a radiation exposure signature consisting of 46 transcripts from a high coverage (40-75 million reads per sample) ONT PromethION PCR cDNA dataset of human blood cells.
In this study, we performed both gene-and isoform-level analyses of two nanopore long-read transcriptome sequencing datasets that follow a simple replicated experimental design: a synthetic 'sequins' (19) PCR-cDNA dataset, and a mouse neural stem cell direct-cDNA dataset. We obtained meaningful results using an analysis pipeline that mostly comprised of 'off-the-shelf' methods developed for shortread data, despite our datasets having only a few million long reads per sample. We found our results for the common two-group experimental design to be reliable in that they are broadly consistent with the available ground-truth or findings from a previous short-read experiment. We found existing methods for isoform identification from long-read data to be unreliable, and introduce a novel method, FLAMES, as part of our isoform-level analysis pipeline.

Study design
Mouse neural stem cells (NSCs) from 4 wild-type (WT) and 3 MommeD1 mutated (Smchd1-null) (20) samples were prepared and sequenced, together with 3 'other' samples from a different experiment. Samples were sequenced in two batches, each containing 6 samples. One WT and one 'other' sample were sequenced in both batches as technical replicates in order to obtain additional reads for these samples.
Technical replicates of synthetic 'sequin' RNA standards (19) from two mixes (A and B) were prepared and sequenced. These samples contain the same transcripts but at variable molar ratios to simulate biological differences in gene expression and alternative splicing. Among the 76 synthetic genes, 21 were up-regulated and 23 were downregulated in mix B compared to mix A. The corresponding transcripts of 28 genes were expressed at different proportions between the two mixes, resulting in DTU for 62 out of 160 transcripts. A further two sequin mix A and two mix B samples were sequenced using Illumina short-read sequencing technology together with RNA from human lung adenocarcinoma cell lines.

Biological materials
Synthetic 'sequin' RNA standards were obtained from the Garvan Institute of Medical Research.

Nanopore sequencing and data preprocessing
Sequin cDNA libraries were constructed with SQK-PCS109 cDNA-PCR sequencing and SQK-PBK004 PCR Barcoding kits using the supplied protocol. Briefly, duplicate libraries of each mix (A1, A2, B1 and B2) were constructed using 15 ng as input for cDNA synthesis. Samples were barcoded 1 to 4 using the supplied PCR barcodes. Transcripts were amplified by 14 cycles of PCR with a 6-min extension time.
Sequencing libraries were individually purified using Beckman Coulter 0.8x AMPure XP beads and quantified using an Invitrogen Qubit 4.0 Fluorometer (ThermoFisher Scientific). Equimolar amounts of each sample were pooled to a total of approximately 160 fmol (assuming median transcript size is 1 kb), and quality control of the pooled library was performed using Agilent Technologies TapeStation 4200. The final library was loaded onto an R9.4.1 Min-ION flow cell and sequenced for 65 h with a buffer refuel at 24 h (using 250 ml buffer FB) using the ONT GridION platform. The fast5 files were base-called by Guppy version 4.0.11 using configuration file dna r9.4.1 450bps hac.cfg to obtain fastq files, trim adaptor sequences and demultiplex barcoded reads. Guppy is only available to ONT customers via the community site (https://community.nanoporetech. com/).
For the NSC dataset we prepared direct-cDNA libraries from 40 ng purified polyadenylated RNA. We combined the ONT direct-cDNA sequencing (SQK-DCS108) protocol (version DCB 9036 v108 revG 30Jun2017) with the one-pot native barcoding protocol (http://lab.loman.net/ protocols/) with extended incubation times (using SQK-LSK109 and EXP-NBD103 kits) for library preparation of the first batch, and used the updated kits SQK-DCS109 and EXP-NBD114 for the second batch (protocol PDCB 9093 v109 revA 04Feb2019). We loaded 100 ng of the final libraries on one PromethION flow cell (FLO-PRO002) per batch. The fast5 files were basecalled by Guppy version 4.0.11 using configuration file dna r9.4.1 450bps hac prom.cfg to yield fastq files. Guppy was also used to trim adaptor sequences from reads and demultiplex barcoded reads. The 'other' samples were removed in downstream analysis. For an overview of our analysis pipeline see Figure 1A.

Illumina sequencing
About 6 ng of sequins mix A or B were added to 1 g of total RNA from human lung adenocarcinoma cell line NCI-H1975 (mix A) and HCC827 (mix B). The pooled RNA was subjected to the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490) and NEBNext Ultra II Directional RNA Library Prep (E7760) for Illumina following manufacturer's instructions and default fragmentation conditions (15 min at 94 • C), 5-fold adaptor dilution and a final amplification of nine PCR cycles. Indexed and pooled libraries were sequenced on a NextSeq500 (Illumina) High Output, 81 cycles paired-end.

Genomic alignment
The NSC reads were aligned to mouse mm10 genome using minimap2 version 2.17-r943-dirty (22) to get bam files. The genome alignments were performed with the arguments -ax splice -uf -k14 -junc-bed, allowing spliced alignments on the forward transcript strand to map with higher sensitivity. It also uses annotated splice junctions to improve the accuracy of mapping at junctions. Gencode release M23 (GRCm38.p6) annotation (23) was used to provide information on known splicing junctions. The sequins ONT reads were mapped to the artificial chromosome chrIS R using minimap2 with the arguments -ax splice -MD. The bam files were sorted and indexed using samtools version 1.6 (24). The sequin Illumina reads were mapped to chrIS R using Subread version 1.6.3 (25).

Gene abundance estimation
Mapped reads were assigned to individual genes and counted by the featureCounts (26) function in the R (https:// www.R-project.org/) Bioconductor (27) package Rsubread version 1.34.4 (25,28). We used the Gencode release M23 (GRCm38.p6) annotation for the NSC data, and sequins annotation GTF file version 2.4 for the sequins data. Arguments isLongRead=TRUE (as recommended in the fea-tureCounts help page when dealing with Nanopore data) and primaryOnly=TRUE to count primary alignments only were specified.

Differential gene expression analysis
Genes in the NSC dataset were annotated using the R/Bioconductor package Mus.musculus (https://bioconductor.org/packages/Mus.musculus/ version 1.3.1) and read counts from technical replicates of the same sample run across different batches were combined (i.e. summed together). For all datasets, we organized and preprocessed the count data using the R/Bioconductor package edgeR version 3.26.8 (18,29). Lowly expressed genes were removed using the filterByExpr function with default arguments. Normalization factors were calculated using the trimmed mean of M-values method (30). Differential gene expression (DGE) analysis was performed using the limma-voom pipeline version 3.40.6 (10,31,32), with sample-specific quality weights (33). Linear models were fitted with either genotype or sequin mix information to create the design matrix, followed by empirical Bayes moderation of t-statistics (34). Raw P-values were adjusted for multiple testing (35).

Mouse NSC short-read data
We obtained DGE results from a previous Illumina shortread RNA-seq study comparing mouse Smchd1-null and WT NSC samples (21,36) available at http://bioinf.wehi. edu.au/folders/smchd1/ and from GEO (accession number GSE65747). Using a limma-voom pipeline, the study reported 1197 differentially expressed (DE) genes (adjusted P-value cutoff of 0.01). We further restricted this list (adjusted P-value cutoff of 0.0001) to give us 218 up-and 54 down-regulated genes in Smchd1-null samples when compared to WT samples. This cutoff resulted in similar numbers of significant genes between the short-and long-read datasets. The DE genes were compared to that of the NSC long-read data using ROAST gene set testing (37) with 9999 rotations.

Transcript-level analysis
We used three different tools to perform isoform detection and quantification: FLAIR version 1.5 (commit 2f8df51) (6) and TALON version 5.0 (commit c41b9dc) (5) for the sequins data, and FLAMES version 0.1.0 (available at https://github.com/XueyiDong/FLAMES) (38) for both sequins and NSC datasets. Default parameters were used to run FLAIR. TranscriptClean (39) version 1.02 which performs reference-based error correction was applied prior to running TALON version 5.0. Transcripts identified by TALON were filtered using default setting.
FLAMES (short for 'Full-Length trAnscript quantification, Mutation and Splicing analysis') is a novel method and software tool developed for long-read RNA-seq data (38), available at https://github.com/LuyiTian/FLAMES. It requires sorted bam files with reads aligned to the genome as input. FLAMES first summarizes the alignment results by grouping reads with similar splice junctions to get a raw isoform annotation. The raw isoform annotation is compared against the reference annotation to correct potential splice site and transcript start/end errors. Transcripts that have similar splice junctions (<5 bp by default) and transcript start/end (<100 bp by default) to the reference tran- script are merged with the reference. High-confidence isoforms are those identified with at least 10 supporting reads. This process will also collapse isoforms that are likely to be truncated transcripts, which are identified as having incomplete 3' regions compared to the full-length forms. A draft transcript assembly is generated at this stage, and all reads are re-aligned to both the known and assembled transcripts and quantified. For reads that align to multiple transcripts, both the proportion of transcript covered by the read alignment and the percentage of read sequence that aligns to each transcript are considered, with the read assigned to the transcript with highest coverage and >80% of its sequence aligned. A configuration file (JSON format) is used to set all parameters mentioned above so that different parameters can used to adapt to different situations. For example, one could set more stringent parameters for merging splice junctions (i.e. decreasing the default from 5 to 1 bp) to better suit long-read data generated by the PacBio platform, which generally has a lower error rate. After running each isoform detection tool, SQANTI (40) version 1.2 was used to classify identified isoforms into structural categories by comparing them to the annotation. An isoform matching a reference transcript can be categorized as 'full splice match' or 'incomplete splice match', based on whether all splice junctions are matched. 'Novel in catalog' is the category for novel isoforms of known genes containing new combinations of already annotated junctions or novel splice junctions composed of annotated donors and acceptors. 'Novel not in catalog' stands for the novel isoforms with novel donors and/or acceptors. Other isoforms can be categorized as either 'antisense', 'fusion' or 'genic/intergenic' isoforms. For both Illumina shortread datasets, Salmon version 1.3.0 (41) 'mapping-based mode' was used to obtain quantification of isoforms identified in corresponding ONT long-read data by FLAMES. Lowly expressed genes and transcripts were removed from downstream analysis using the dmFilter function from the DRIMSeq package. For all long-and short-read datasets, genes were required to have an associated gene count (obtained by summing counts across all transcripts for a given gene) of 10 or more in every sample. A second filter required transcripts to have 10 or more counts in at least 3 samples in the NSC data, and in at least 2 samples in the sequins data.
DTU analysis was performed using two methods: the R/Bioconductor package DRIMSeq version 1.12.0 (9), and diffSplice from the limma package version 3.40.6. Originally, DRIMSeq was designed for use with transcriptlevel counts in short-read data, giving adjusted P-values at both the gene-level and feature-level (transcripts). Diff-Splice analyses exon-level counts in short-read data to indirectly call for differences in isoform proportions, and reports adjusted P-values at the gene-level (Simes adjustment and/or F-tests) and exon-level (t-tests). For long-read data, we applied the diffSplice to transcript-level counts rather than exon-level counts as carried out by Love et al. (11). Additionally, the stage-wise method from R/Bioconductor package stageR version 1.6.0 (12) was also applied to the raw P-values from DRIMSeq (gene-and transcript-level) and diffSplice (Simes and t-tests) methods for FDR control to give stageR gene-and transcript-level adjusted P-values.

Data and code availability
RNA-seq data can be accessed from Gene Expression Omnibus (GEO) under accession numbers GSE151984 (sequins long-read), GSE151841 (NSC long-read data) and GSE164598 (sequins short-read). All code used to perform these analyses are available from https://github.com/ XueyiDong/LongReadRNA.

Data quality
To assess the quality of our long-read datasets, raw long reads were pre-processed and assigned to gene-level counts using an appropriate reference genome. Figure 1B shows the number of reads (or amount of information) retained after some crucial steps in processing the NSC data. A total of ∼81 million reads were successfully sequenced and base-called. Approximately 70%, or ∼57 million reads had an average base quality score >7 and passed quality filtering. Around 51 million quality-passed reads were detected with adaptor and barcode sequences in the trimming and demultiplexing steps. The reads from our samples of interest were then mapped to the genome and assigned to genes, producing ∼33 million gene-level counts. For the sequins dataset, ∼11 million raw reads yielded ∼6.4 million gene-level counts (Supplementary Figure S1). In comparison, the sequins short-read dataset had ∼162 million genelevel counts.
In the NSC dataset, median read length is 752 bases. Figure 1C shows that shorter reads tend to have higher median read quality, but the difference is subtle. The quality of 'extra long' reads (≥3000 bases) were similar to reads of other length categories, indicating Nanopore's ability to detect transcripts in this size range. A small proportion of reads (∼1%) exceed 5 kilobases. Similar to the NSC dataset, the sequins dataset had a median read length of 716 bases, which is shorter than its expected value of 908 bases. The library size (sum of gene counts) of samples in the NSC dataset varied between 2.7 and 7.7 million reads ( Figure  1D). In comparison, the library size of samples in the shortread NSC study (21) range between 18.6 and 23.2 million reads.

Gene expression analysis
In short-read RNA-seq, transcripts (or genes) are fragmented for sequencing, such that longer transcripts can be over-represented relative to transcripts that are shorter. As a consequence, DGE analyses are biased towards the detection of genes (or transcripts) that are relatively long (42). Also, DGE analyses may be confounded by DTU such that gene-level counts are affected by the varying proportions of transcripts with varying lengths. One advantage to longread RNA-seq protocols is that they do not include the fragmentation step, and should theoretically be unbiased to gene length. To examine this, we looked at the relationship between gene length and gene expression using log 2 -countsper-million (log-CPM) values. Gene length is weakly associated with expression in both long-read datasets; Pearson correlation of 0.035 for the NSC dataset ( Figure 1E) Figure S2), whereas the equivalent correlation in the short-read NSC study (21) is greater at 0.20.
We applied the limma-voom workflow designed for shortread DGE analysis to the long-read data. We first analysed the sequins data to check whether the approach was appropriate using the ground-truth available. The analysis was carried out using voomWithQualityWeights to account for sample-level heterogeneity by estimating samplespecific quality weights based on similarity of gene expression within the same group. The sample weights were combined with voom precision weights that are based on the mean-variance relationship estimated from the data. Even though there were only 69 genes present in the dataset (as opposed to tens of thousands in a typical dataset), the mean-variance trend observed for the sequins data (Supplementary Figure S3) was similar to that of short-read RNAseq data (31).
Linear modelling on the gene-level counts were carried to obtain estimated log 2 fold-change (logFC) values between mix A and B. Estimated values were highly correlated (R 2 = 0.934) with expected logFCs (Figure 2A). Using an adjusted P-value cutoff of 0.05, 21 down-regulated and 18 upregulated genes were detected between mix B and A. There were no false discoveries, and only 2 of the true differentially expressed genes were not detected.
The same DGE analysis workflow was also applied to the short-read sequins data, with 2 false negative and 3 false positive discoveries made. Upon checking the annotation, we found that all of false positive genes in the short-read data were long (>2400 bp) DTU genes that were lowly expressed (abundance ≤ 0.24; median abundance among all genes = 7.55). The t-statistics from DGE analysis of shortread data were observed to be highly correlated (R 2 = 0.838) with the t-statistics from the long-read data ( Figure 2B). Overall, results from the sequin synthetic control data indicate that the limma-voom pipeline is powerful and reliable when applied to long-read data, so we next applied it to the NSC dataset.
Unsupervised clustering by multidimensional scaling (MDS) was used to observe the relationships between NSC samples. Dimension 1 in the MDS plot roughly separates samples by genotype ( Figure 2C), although a Smchd1-null sample (sample 7) is positioned more closely to WT samples. The mean-variance trend for this dataset is again typical of what is observed in short-read RNA-seq experiments (31) ( Figure 2D). Estimated sample weights favoured samples that distinguished groups across dimension 2 of the MDS plot, giving samples 2 and 3 in the Smchd1-null group weights that are >1, as well as samples 1 and 5 in the WT group (Supplementary Figure S4). Using the default adjusted P-value cutoff of 0.05, only 12 genes were detected as DE. Using a more liberal adjusted P-value cutoff of 0.25 to account for the small library sizes, detected 81 downregulated and 63 up-regulated genes between Smchd1-null and WT samples ( Figure 2E). The Smchd1 gene, which was depleted in Smchd1-null samples, was detected as the most significantly down-regulated gene in the comparison (highlighted in Figure 2E) and serves as a positive control for this analysis.
In a previous short-read study on the same mouse NSC groups (21,36), the imprinted genes Ndn, Mkrn3 and Peg12 were reported as up-regulated. These genes were also found to be DE in the long-read dataset (highlighted in Figure 2E). Further comparison between the short-and longread datasets was carried out using a barcode plot (Figure 2F). The barcode plot shows that genes that were upregulated in the short-read dataset (red vertical lines in the plot) also tend to be up-regulated in our long-read dataset (positioned towards the right of the plot). Specifically, the genes that were most highly up-regulated in the short-read dataset as ranked by logFC (long red vertical lines), are also highly up-regulated in the long-read data (further right in the plot). The same goes for down-regulated genes in the short-read dataset (blue vertical lines in the plot), which tend to be down-regulated in the long-read dataset (positioned towards the left of the plot). We tested concordance of the datasets formally by applying the ROAST gene set testing method (37) to our long-read data. Using both upand down-regulated gene sets from the short-read dataset, weighted on their logFC values, ROAST returned an 'up' Pvalue of 0.086, which indicates that transcriptional changes for the comparison of Smchd1-null versus WT are somewhat consistent between the two datasets (up-regulated genes in the short-read data tend to be up-regulated in the long-read data, and down-regulated genes in the long-read data tend to be down-regulated in the long-read data). The relatively large ROAST P-value and overall lack of power to detect differentially expressed genes is likely due to relatively low sequencing levels per sample and within-genotype sample heterogeneity in the long-read experiment. Smoothed scatter plots of the logFCs and t-statistic for each gene between the short-and long-read datasets are presented in Supplementary Figure S5. Discordance between the longand short-read data is likely caused by the fact that samples were not perfectly matched. The short-read experiment, which was performed a few years earlier, used samples derived from different animals which may have been at a slightly different developmental stage compared to the newer samples profiled using long-read RNA-seq. In addition, since these samples are from primary cells in culture, the number of passages post derivation can also influence gene expression and any subsequent differential expression results.

Transcript-level analysis
Transcript-level analysis of nanopore RNA-seq data usually starts with isoform detection. To test which tool is best suited to nanopore data, we compared two popular tools, FLAIR and TALON with our novel FLAMES pipeline on the sequins dataset. Ideally, all transcripts that appear in the sequins annotation should be detected, and there should be no novel isoforms. Our results showed that FLAMES detected the most sequin transcripts ( Figure 3A, 'full splice match' category) and fewer artefactual isoforms ( Figure  3A, other categories). While most sequin transcripts were also detected by FLAIR, a large number of artefactual isoforms were also identified, especially those classified as 'novel in catalog' for which we know there should be none. TALON detected ∼77% of the sequin transcripts, and a disproportionately large number of artefactual isoforms, especially in the 'antisense' and 'novel not in catalog' categories. When we looked into the number of reads assigned to transcripts in each category ( Figure 3B), the majority of counts in FLAMES were from known isoforms, while more than half of the counts in FLAIR and TALON were from artefactual isoforms. The total number of read counts from FLAMES (∼4.9 M) and TALON (∼4.2 M) are similar, while FLAIR recovered a lower number (∼2.7 M). Results from the sequins dataset indicated that the novel FLAMES pipeline outperformed the other two methods.
To further assess the performance of FLAMES and the quality of the dataset, we calculated the coverage fraction of transcripts by individual reads. Here, reads covering 95% or more of the bases of their corresponding transcript are defined as 'full-length'. In our sequins data, 47% of reads were found to be full-length, which is similar to Gleeson et al (14)'s ONT MinION direct RNA dataset. Reads assigned to longer transcripts are less likely to be full-length ( Figure 3C), consistent with findings from Jenjaroenpun et al. (16). This suggests that some reads may be truncated in our sequins dataset, which presumably occurs during library preparation. The truncated reads may have resulted in the detection of 'incomplete splice match' isoforms (blue in Figure 3A and 3B).
Next, we used DRIMSeq and the diffSplice function in limma for DTU analysis of long-read data. We also combined the methods with the stage-wise analysis from the stageR (12) package since it was recommended in the DRIMSeq vignette for statistical improvement and enhanced biological interpretation of results. We expect good performance of DTU analyses comparing mixes A and B since the observed CPM values of sequin transcripts were highly correlated with their expected abundances (Pearson correlation = 0.81, Figure 3D). We expect up to 5% of false discoveries by applying at adjusted P-value cutoff of 0.05. diffSplice methods performed better than DRIMSeq overall with no false discoveries and high true positive rate (TPR > 0.82) ( Figure 3E and 3F, Supplementary Figure S6). DRIM-Seq had comparable TPR, but did not properly control the FDR, such that more false discoveries were found (FDR between 0.08 and 0.10) than expected. For transcript-level testing, stageR improved the TPR of both methods whilst maintaining the same FDR. However, stageR did not improve the performance in gene-level testing for either of the two methods (i.e. the TPRs were unchanged while the FDR increased for DRIMSeq by 0.005. We also quantified the expression of the same transcripts and performed DTU analysis on short-read sequins data. The observed transcripts per kilobase million (TPM) values were also highly correlated with their expected abundances (Pearson correlation = 0.86, Supplementary Figure  S7). However, the FDR of all DTU tests were higher than that of long-read data, and were not properly controlled (FDRs ranged between 0.10 and 0.17 using an adjusted Pvalue cutoff of 0.05, Supplementary Figure S8). The higher error rates in short-read data suggests that both methods performed better on long-read data than on the short-read data that they were originally designed for use on. We examined the concordance between the datasets by comparing the top n most significant DTU genes. For both DRIM-Seq and diffSplice, in the top 19 genes, which is the number of true DTU genes in the datasets, there were 16 genes in common between the two methods ( Supplementary Figure S9). We demonstrate that our pipeline and combination of methods for transcript-level analysis produces accurate transcript quantification and identification of DTU, and provides confidence for application to other long-read transcriptomic datasets.
We then applied our transcript-level analysis workflow to the NSC dataset. The FLAMES pipeline returned 38 857 unique isoforms from 9837 genes, of which 38% were classified as novel (Supplementary Figure S10), which is a lot more than what was observed in the sequins dataset. Since we observed a very low level of falsely discovered isoforms in the sequins data, we assume that that most of these novel isoforms are real, which suggests that the current mouse transcript annotation is incomplete. Of the mapped reads, 32.3% were assigned to novel isoforms, the majority of which were from the 'novel not in catalog' category (Supplementary Figure S11). Using an adjusted P-value cutoff of 0.05, DRIMSeq found one gene Pisd as having DTU in the Smchd1-null versus WT comparison, while diffSplice did not detect any DTU genes, although Pisd had the smallest P-value (raw P-value = 0.0002) among all the genes tested. Transcript ENSMUST00000201980.4 ('Known5') and EN-SMUSG00000023452.19 32736305 32746312 1 ('Novel2') in Pisd was identified by DRIMSeq to have differential usage between the two groups (Supplementary Figures S12 and S13).
We also performed DTU analysis on the NSC short-read data. Based on the sequins results, DTU analysis on short reads may be less reliable, with more false discoveries than expected. Using an adjusted P-value cutoff of 0.05, DRIM-Seq found 139 DTU genes, while diffSplice only found 9 DTU genes (Supplementary Figure S14). There were three DTU genes (Pisd, Cyth2 and Pabpn1) found in common by both methods. Transcript ENSMUST00000201980.4 in Pisd was identified by both methods to have a higher usage in Smchd1-null samples than in WT samples (Supplementary Figure S15, 'Known5'), which was concordant with what was observed in the long-read analysis. Since we observed high TPRs in our DTU analysis for the sequins short-read data and the library size of the NSC short-read dataset is relatively high, we expect our analysis to have sufficient power to detect DTU genes if they were present in this dataset.

DISCUSSION
Our DGE analysis uses a limma-voom workflow and shows that results from PCR-cDNA and direct-cDNA long-reads are reliable, such that estimated results are comparable to the known truth in the sequins synthetic control dataset, and concordant with corresponding short-read studies. Although the total library size in the sequins dataset is lower than that of the NSC dataset, more reads were assigned per gene since the dataset contains a small set of genes, which improved power for DGE analysis. Overall, comparisons using long-read experiments suffer from a lack of statistical power due to low library sizes. It would be desirable for long-read transcriptomic studies to have total read numbers that are more comparable to what is routinely achieved in short-read experiments (20-50 million reads per sample is not unusual). We expect this to occur in the near future as throughput of long-read experiments increases.
We also looked into transcript-level analysis of long-read data and found our novel FLAMES pipeline to be reliable in both isoform detection and quantification. The high false positive rate of FLAIR and TALON for isoform detection suggests that these algorithms need further improvement to adapt to the high error rates in long-read sequencing. Despite methods being designed originally for shortread data, diffSplice (with or without stageR) performed very well in DTU analyses of the sequins long-read data using transcript-level counts, with better FDR control observed in long-read DTU analysis than in the short-read DTU analysis. We believe these methods could be applied to other datasets with confidence, but may lack power to detect DTU genes if transcript counts are very low. Notably, relative to DGE analyses, a DTU analysis further splits genelevel counts into associated isoforms which reduces power for statistical testing. For this reason, the power to detect DTU genes in the NSC long-read dataset is reduced relative to the sequins dataset since the latter contains far fewer ex-pressed genes and transcripts to begin with, such that transcripts have higher counts on average. Another potential issue in the application of these methods to our NSC dataset is that altered expression of the gene Smchd1 may not affect RNA splicing mechanisms, which could mean there is a biological explanation for why very little DTU was observed in these data across both the short and long-read analyses.
Our study is the first to test a pipeline for both genelevel DGE analysis and transcript-level DTU analysis of nanopore long-read RNA-seq data. Whilst the sequencing depth is relatively low, we are still able to obtain reasonable results using pre-existing methods designed for short reads, namely the limma software. We expect that other short-read tools such as edgeR and DESeq2 may also be appropriate, as used in other studies (14,17) although further benchmarking efforts are required to confirm this. Exploring the strengths and weaknesses of different analysis methods on data arising from both the Nanopore and PacBio long-read platforms using a specially designed benchmarking dataset is planned as future work.
We hope that our analysis will encourage further research into the potential for long-read RNA-seq to be used in place of short-read RNA-seq, allowing for the simultaneous exploration of gene-level and isoform-level changes within the same experiment in a more comprehensive way.

SUPPLEMENTARY DATA
Supplementary Data are available at NARGAB Online. Conflict of interest statement. MBC and RDP have received support from Oxford Nanopore Technologies (ONT) to present their findings at scientific conferences. However, ONT played no role in study design, execution, analysis or publication.