ANCHOR: haplotype-aware allelic and isoform inference from single-cell long-read RNA sequencing with de novo variant calling

Abstract

Long-read RNA sequencing enables haplotype- and isoform-resolved allelic analysis of transcriptomes, yet extending this capability to single cells and distinct cell types remains computationally challenging due to sparse coverage, sequencing errors, incomplete variant information, and reference-biased transcript assignment. Here we present ANCHOR, a haplotype-aware framework for single-cell long-read RNA sequencing that performs de novo expressed-variant discovery, molecule-level haplotype assignment and isoform-resolved allelic quantification. ANCHOR combines a signed-graph variant caller, pair hidden Markov modelling and beta-binomial UMI aggregation to infer parental allele counts for genes and splice-resolved isoforms, without requiring a pre-existing phased genotype or deep learning. In human single-cell long-read RNA benchmarks, ANCHOR improved variant-calling performance over tested long-read RNA callers at single-cell and low-to-moderate coverage, and its beta-binomial model reduced depth-driven false positives in allele-specific expression testing. Applied to newly generated single-cell long-read RNA-seq data from reciprocal mouse crosses during gastrulation, ANCHOR resolved cell-type- and isoform-specific parent-of-origin imprinting and identified an antagonistic maternally biased Sgce isoform. ANCHOR provides a general framework for allele-and isoform-resolved analysis of diploid single-cell long-read transcriptomes.