Abstract
Long-read RNA sequencing is a powerful technology to link transcript structures to genetic variants, but this type of analysis is not often performed owing to the lack of end-user tools. Here we introduce longcallR for joint single-nucleotide polymorphism calling, haplotype phasing and allele-specific analysis, which achieves high accuracy on benchmark datasets. Applied to 202 human samples, longcallR identified 88 significant allele-specific splicing events per sample on average, of which 46% involved unannotated junctions.
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Data availability
The raw reads for HG002a:MAS:cDNA are available at https://downloads.pacbcloud.com/public/dataset/Kinnex-full-length-RNA/DATA-Revio-HG002-1/. The raw reads for HG002b:MAS:cDNA, HG002c:MAS:cDNA, HG002d:MAS:cDNA, HG004:MAS:cDNA, HG005:MAS:cDNA, HG002a:ISO:cDNA, HG002b:ISO:cDNA, HG002c:ISO:cDNA, HG004:ISO:cDNA and HG005:ISO:cDNA can be accessed at https://ftp-trace.ncbi.nlm.nih.gov/giab/ftp/data_RNAseq/. The WTC11:ONT:cDNA reads can be found in ENCODE under ENCSR539ZXJ. The raw reads for HG002a:ONT:cDNA, HG002b:ONT:cDNA, HG004:ONT:cDNA and HG005:ONT:cDNA are available at https://s3.amazonaws.com/gtl-public-data/giab/bams/cDNA/05_09_23_R941_GIAB_cDNA_PCS111_NA26105_Guppy_6.4.6_sup.pass.fastq.gz.hg38.bam, https://s3.amazonaws.com/gtl-public-data/giab/bams/cDNA/05_09_23_R941_GIAB_cDNA_PCS111_NA27730_Guppy_6.4.6_sup.pass.fastq.gz.hg38.bam, https://s3.amazonaws.com/gtl-public-data/giab/bams/cDNA/05_09_23_R941_GIAB_cDNA_PCS111_NA24143_Guppy_6.4.6_sup.pass.fastq.gz.hg38.bam and https://s3.amazonaws.com/gtl-public-data/giab/bams/cDNA/05_09_23_R941_GIAB_cDNA_PCS111_NA24631_Guppy_6.4.6_sup.pass.fastq.gz.hg38.bam. The HG002:ONT:dRNA, HG004:ONT:dRNA and HG005:ONT:dRNA datasets, generated by the University of Hong Kong, are available from the NCBI under SRX26304755, SRX26304756 and SRX26304757. HPRC human samples are available at https://s3-us-west-2.amazonaws.com/human-pangenomics/index.html?prefix=working/HPRC/. For ground-truth DNA variants, the VCF files for HG002, HG004 and HG005 are available at GIAB v4.2.1. The VCF for WTC11 is provided by the Allen Institute at https://open.quiltdata.com/b/allencell/tree/aics/wtc11_short_read_genome_sequence/. The reference genome GRCh38 can be downloaded from NCBI. All generated SNP calls, ASJs and underlying data for the figures are available via Zenodo at https://doi.org/10.5281/zenodo.17842979 (ref. 27). Source data are provided with this paper.
Code availability
LongcallR is available via GitHub at https://github.com/huangnengCSU/longcallR. LongcallR-nn is available via GitHub at https://github.com/huangnengCSU/longcallR-nn. LongcallR scripts are available via GitHub at https://github.com/huangnengCSU/longcallR_scripts. The Nextflow of longcallR is available via GitHub at https://github.com/huangnengCSU/longcallR-nf.
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Acknowledgements
This work is supported by National Institute of Health (grant nos. R01HG010040, R01HG014175, U24CA294203, U01HG013748 and U41HG010972 (to H.L.)). We thank the National Genome Research Institute for funding the following grants supporting the creation of the human pangenome reference: U41HG010972, U01HG010971, U01HG013760, U01HG013755, U01HG013748, U01HG013744 and R01HG011274, and the HPRC (BioProject ID: PRJNA730823).
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N.H. and H.L. designed the algorithm, implemented longcallR and drafted the paper. N.H. conducted the performance evaluation of longcallR for SNP calling, haplotype phasing and allele-specific analysis. The HPRC provided the 202 MAS-seq human sample data.
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Nature Methods thanks Andrey Prjibelski and the other, anonymous, reviewer(s) for their contribution to the peer review of this work. Peer reviewer reports are available. Primary Handling Editors: Lei Tang and Lin Tang, in collaboration with the Nature Methods team.
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Extended data
Extended Data Fig. 1 Accuracy of SNP calling and haplotype phasing.
A, precision and recall for SNP calling on PacBio datasets between longcallR (circles) and Clair3-RNA (triangles). Colors represent different samples. Genomic SNPs from GIAB are taken as the ground truth. B, precision and recall for SNP calling on Nanopore datasets. C, Phasing switch error rates between longcallR-phase (green) and WhatsHap (yellow) across PacBio and Nanopore datasets. Trio-based HG002 SNP phasing from GIAB is taken as the ground truth. Lower switch error rates indicate higher phasing accuracy. D, Phasing hamming error rates in haplotype phasing.
Supplementary information
Supplementary Information (download PDF )
Supplementary Tables 1–15, Figs. 1–12 and Notes 1–4.
Source data
Source Data Fig. 2 (download XLSX )
Upset plot source data, Venn plot source data, cumulative count of significant ASJ genes across HPRC samples, count of known and novel ASJ events across HPRC samples.
Source Data Extended Data Fig. 1 (download XLSX )
Precision and recall for SNP calling on PacBio datasets between longcallR and Clair3-RNA, precision and recall for SNP calling on Nanopore datasets, phasing switch error rates between longcallR-phase and WhatsHap across PacBio and Nanopore datasets, phasing hamming error rates between longcallR-phase and WhatsHap across PacBio and Nanopore datasets.
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Huang, N., Li, H. & Human Pangenome Reference Consortium. SNP calling, haplotype phasing and allele-specific analysis with long RNA-seq reads. Nat Methods (2026). https://doi.org/10.1038/s41592-026-03045-6
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DOI: https://doi.org/10.1038/s41592-026-03045-6
