Pre-Assembly NGS Correction of ONT Reads Achieves HiFi-Level Assembly Quality

Abstract

Recently developed hybrid assemblies can achieve Telomere-to-Telomere (T2T) completeness of some chromosomes. However, such approaches involve sequencing a large volume of both Pacific Biosciences high-fidelity (HiFi) and Oxford Nanopore Technologies (ONT) sequencing reads. Along with this, third-generation sequencing techniques are rapidly advancing, increasing the available length and accuracy. To reduce the final cost of genome assembly, here we investigated the possibility of assembly from low-coverage samples and with only ONT corrected by Next-Generation Sequencing (NGS) sequencing reads. We demonstrated that ONT-based assembly approaches corrected by NGS can achieve performance metrics comparable to more expensive hybrid approaches based on HiFi sequencing. We investigated the assembly of different chromosomes and the low-coverage performance of state-of-the-art hybrid assembly tools, including Verkko and Hifiasm, as well as ONT-based assemblers such as Shasta and Flye. We rigorously evaluated the performance of MGI, Illumina, and stLFR NGS technologies across various aspects of hybrid genome assembly, including pre-assembly correction, haplotype phasing, and polishing, and found them to be similarly effective. Additionally, we proposed two-round assembly methods that utilize stLFR linked-read data to achieve assembly phasing performance comparable to that obtained with trio data.