The accuracy of methods for assembling transcripts from short-read RNA sequencing data is limited by the lack of long-range information. Here we introduce Ladder-seq, an approach that separates transcripts according to their lengths before sequencing and uses the additional information to improve the quantification and assembly of transcripts. Using simulated data, we show that a kallisto algorithm extended to process Ladder-seq data quantifies transcripts of complex genes with substantially higher accuracy than conventional kallisto. For reference-based assembly, a tailored scheme based on the StringTie2 algorithm reconstructs a single transcript with 30.8% higher precision than its conventional counterpart and is more than 30% more sensitive for complex genes. For de novo assembly, a similar scheme based on the Trinity algorithm correctly assembles 78% more transcripts than conventional Trinity while improving precision by 78%. In experimental data, Ladder-seq reveals 40% more genes harboring isoform switches compared to conventional RNA sequencing and unveils widespread changes in isoform usage upon m6A depletion by Mettl14 knockout.
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Ladder-seq raw sequencing data from WT and Mettl14 KO mouse NPCs, conventional RNA-seq data from WT mouse NPCs and ONT long-read sequencing from WT and Mettl14 KO mouse NPCs are available in the Gene Expression Omnibus (GSE158985). m6A peaks from two replicates of m6A sequencing in mouse NPCs were downloaded from the Gene Expression Omnibus (GSE104686).
The kallisto-ls, StringTie-ls and Trinity-ls programs and workflows are available at:
respectively. The results of our benchmark studies can be reproduced via a Snakefile33, available at https://github.com/canzarlab/ladderseq_benchmark.
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We would like to thank A. A. Kha for helpful discussions and the members of the Canzar laboratory for comments and suggestions. We thank P. J. Shaw and M. Halic for constructive feedback on mRNA length separation. We thank S. Esser for excellent technical assistance. S. Chakraborty was supported by a Deutsche Forschungsgemeinschaft (DFG) fellowship through the Graduate School of Quantitative Biosciences Munich. F.R.R. was supported, in part, by the Bavarian Gender Equality Grant. This work was funded by the TTIC Research fund (to S. Canzar) and by grants from the National Institutes of Health (R35NS097370 to G.M. and R35NS116843 to H.S), the Simons Foundation (575050 to H.S.), the Deutsche Forschungsgemeinschaft (DFG, German Research Foundation) SFB-TRR 338/1 2021-452881907 (to S. Canzar), DFG Sachbeihilfe (417912216 to M.L.S.), Deutsche Gesellschaft für Muskelkranke e.V. Forschungsförderung (awarded in 2019 to M.L.S.), the Institute for Basic Science (IBS-R008-D1 to A.H. and H.C.), the National Research Foundation of Korea (2019R1C1C1006600 and 2020M3A9E4039670 to K.-J.Y. and 2021R1C1C1010758 to A.H. and H.C.), funded by the Ministry of Science, and by ICT of Korea, Young Investigator Grant, from the Suh Kyungbae Foundation (to K.-J.Y.).
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Extended Data Fig. 1 Reduced (effective) gene complexity in Ladder-seq.
We estimate transcript expression in Mettl14 KO sample 1 using kallisto on Ladder-seq reads pooled across bands and show the histogram of gene complexity measured as the number of expressed transcripts. In Ladder-seq, we partition the set of expressed transcripts into 7 bands and count the number of transcripts contained in each band according to their annotated length (plus 200 nt average poly(A) tail size46), assuming cuts at 1000 bp, 1500 bp, 2000 bp, 3000 bp, 4000 bp and 6000 bp. The resulting histogram of effective gene complexity shows an increased fraction of gene bands with low complexity.
Extended Data Fig. 2 In silico gel for WT and KO NPC samples.
For every annotated transcript the intensity of a point with y- coordinate equal to its annotated length (plus 200 nt average poly(A) tail size) shows the fraction of reads obtained from each band (x-axis) that can be assigned unambiguously to it. As expected, each band contains predominantly reads from transcripts of a distinct length range.
Extended Data Fig. 3 Overview of the benchmark strategy.
1. The ground truth transcriptome including abundances and error profile is calculated by RSEM from GEUVADIS sample NA12716_7. 2. Reads are simulated from the ground truth transcriptome by RSEM to obtain RNA-seq samples of different sequencing depths. 3. A matching Ladder-seq sample is obtained by separating reads in silico according to probability mass functions estimated from our NPC Ladder-seq sample (and variants thereof). 4. Transcripts are quantified and assembled by our Ladder-seq tailored transcript analysis methods kallisto-ls, StringTie-ls, and Trinity-ls from the Ladder-seq sample, while their conventional counterparts are run on the corresponding RNA-seq sample. 5. The results are compared to the ground truth to evaluate and compare their accuracy.
Extended Data Fig. 4 Quantification accuracy of kallisto-ls on 75 million simulated reads.
Mean values across 20 simulations are reported. Pearson correlation of estimated and ground truth abundance in log2 transformed transcripts per million (TPM) and mean absolute relative difference (MARD) are shown as a function of gene complexity, that is the number of transcripts expressed by a gene. For ease of visualization, we omit genes expressing a single transcript, many of which are estimated to be lowly expressed in GEUVADIS sample NA12716_7 by RSEM.
Extended Data Fig. 5 Accuracy of transcript assembly from 75 million simulated reads.
RNA-seq and Ladder-seq reads were aligned identically to the reference genome (GRCh38) using STAR. Sensitivity and precision of StringTie-ls and its conventional counterpart StringTie2 are shown as a function of gene complexity measured as the number of expressed transcripts. Sensitivity and precision are calculated with respect to the same set of ground truth transcripts as in the smaller 30 million read pairs data set.
Extended Data Fig. 6 Accuracy of de novo transcript assembly from 30 million (top row) and 75 million (bottom row) simulated reads.
(a) Sensitivity of Trinity-ls and its conventional counterpart Trinity at 90% transcript length cut-off is shown as a function of gene complexity measured as the number of expressed transcripts. (b) Total number of correctly assembled transcripts at different transcript length cut-offs. (c) Precision at different transcript length cut-offs.
Extended Data Fig. 7 Ladder-seq improves differential analysis of reconstructed transcriptomes.
(a) Computational pipeline for differential isoform usage analysis with conventional RNA-seq and Ladder-seq. Reads were aligned using STAR aligner prior to transcript assembly for both pipelines. (b) Venn diagram showing overlap between switching genes identified by Ladder-seq and conventional RNA-seq. (c and e) Isoform switches identified only by Ladder- seq in genes Exo1 and Tram1l1 (between n=4 WT and n=4 KO samples). Red arrows show location of m6A methylation. TCONS_00000541 and TCONS_00000542 are novel isoforms of Exo1 detected only by Ladder-seq. TCONS_00006855 is a novel isoform of Tram1l1 that was assembled by both methods, but conventional RNA-seq failed to identify the isoform switch. Without length information, conventional RNA-seq reads in KO bands 2 and 3 were predominantly assigned to the annotated transcript in band 4. Barplots represent mean ± SEM; ***FDR corrected p<0.001. (d and f) Coverage plots for switching genes Exo1 and Tram1l1 showing separation of reads from transcripts of different lengths. (g) Jensen Shannon divergence for Ladder-seq and conventional RNA-seq for all identified transcripts grouped by relative difference in abundance estimation by both methods (n=18761 for <0.5, n=12722 for 0.5-1, n=7918 for 1-1.5, n=6292 for 1.5-2 relative difference). Relative difference is defined as the absolute difference in estimated transcript abundance (in TPM) divided by the average of the two. Boxplot definition: Bottom and top of the box correspond to lower and upper quartiles of the data, bar is the median and whiskers are median ± 1.5x interquartile range.
Extended Data Fig. 8 Mettl14 KO leads to isoform switches in m6A methylated genes.
(a) Gene Ontology for m6A methylated genes containing isoform switches. (b) Isoform switch in Ptprz1 (between n=4 WT and n=4 KO samples). Red arrow shows location of m6A methylation. Barplots represent mean ± SEM; ***FDR corrected p<0.001. (c) Gene Ontology analysis for genes with loss of protein domains in KO NPCs. (d) Splicing analysis: Number of gains and losses of each splicing event in KO NPCs. A3: Alternative 3’ acceptor site; A5: Alternative 5’ acceptor site; ES: Exon skipping; IR: Intron retention; MEE: Mutually exclusive exon; MES: Multiple exon skipping. (e) Gene Ontology enrichment analysis of genes with intron retention loss in KO NPCs.
Extended Data Fig. 9 Comparison of Ladder-seq and ONT-cDNA sequencing on mouse NPCs.
(a) Orange bars show validation by StringTie2 (left panel) or by an independent ONT dataset (Dong et al.) (right panel) of transcripts found by both Ladder-seq and ONT-cDNA while light blue bars show validation values for transcripts reported only by ONT-cDNA. In comparison, 32.5% of transcripts uniquely identified by Ladder-seq (average TPM ≥ 1) were also identified in the dataset by Dong et al. (b) Boxplots showing expression levels (TPM) for transcripts identified both by long- reads and Ladder-seq (green boxes) and for transcripts identified only by Ladder-seq (grey boxes). Left panel shows values for all Ladder-seq transcripts with TPM higher than 1 (n=6169 identified only by Ladder-seq, n=15099 by both). Right panel shows values for Ladder-seq switching transcripts with TPM higher than 1 (n=905 identified only by Ladder-seq, n=2012 by both). Boxplot definition: Bottom and top of the box correspond to lower and upper quartiles of the data, bar is the median and whiskers are median ± 1.5x interquartile range.
Extended Data Fig. 10 Cumulative percentage of Ladder-seq transcripts identified by long-read sequencing.
Bars show percentage of Ladder-seq transcripts identified by FLAIR (green), plus those additionally identified by StringTie2 (blue), plus transcripts additionally found in a recently published long-read mouse NPC transcriptome (light blue).
Supplementary Figs. 1–14 and Tables 1–23
Supplementary Table 19, switching isoforms in Mettl14 KO NPCs.
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Ringeling, F.R., Chakraborty, S., Vissers, C. et al. Partitioning RNAs by length improves transcriptome reconstruction from short-read RNA-seq data. Nat Biotechnol 40, 741–750 (2022). https://doi.org/10.1038/s41587-021-01136-7