A little longer, a lot better: simulation-guided exploration of extended-length single-end barcoded reads for structural variant detection
TL;DR
increasing read length consistently improves SV detection accuracy, with SE1000_stLFR achieving the best performance among the evaluated stLFR configurations and showing competitive performance relative to ICLR- and pangenome-based approaches, while approaching the performance of long-read methods.
Abstract
Abstract Motivation Accurate detection of genetic variants, including single nucleotide polymorphisms (SNPs), small insertions and deletions (INDELs), and structural variants (SVs), is essential for comprehensive genomic analysis. While short-read sequencing performs well for SNP and INDEL detection, it remains limited in resolving SVs, particularly in complex genomic regions, due to its short read length. Linked-read sequencing technologies, such as single-tube Long Fragment Read (stLFR), partially address this limitation by incorporating molecular barcodes to provide long-range information. Results In this study, we evaluate conventional paired-end linked reads (PE100_stLFR) and explore a conceptual extension: long single-end barcoded reads of 500 bp (SE500_stLFR) and 1000 bp (SE1000_stLFR). We developed stLFR-sim, a Python-based simulator that reproduces the stLFR workflow and enables realistic benchmarking. Using a high-quality T2T assembly of HG002, we generated multiple datasets across 12 sequencing configurations. SVs were called using Aquila_stLFR (v2) and benchmarked against the Genome in a Bottle (GIAB) HG002 SV truth set with Truvari. We show that simulated PE100_stLFR has the same trade-off pattern between precision and recall in SV calling compared to real data. Increasing read length consistently improves SV detection accuracy, with SE1000_stLFR achieving the best performance among the evaluated stLFR configurations and showing competitive performance relative to ICLR- and pangenome-based approaches, while approaching the performance of long-read methods. Collectively, our results highlight the potential of extended-length single-end barcoded reads for improving SV detection and demonstrate how simulation can be used to evaluate prospective linked-read sequencing designs. Availability and implementation stLFR-sim is a Python-based, open-source simulator for paired-end and extended-length single-end barcoded sequencing. The software is freely available under the MIT License at https://github.com/maiziezhoulab/stLFRsim.