Over the past 5 years, long-read sequencing (LRS) has significantly advanced genetic variant detection. By accessing repetitive genomic regions and offering precise variant breakpoint data, LRS technologies like those from Oxford Nanopore Technologies (ONT) and PacBio Biosciences (PacBio) have improved structural variant (SV) detection threefold over short-read methods. These improvements enable the generation of reference as well as phased genome assemblies, shedding light on genetic variation’s impact on disease. The authors compared two LRS platforms and shared their results.
In the study, ultra-long Oxford Nanopore Technologies (UL-ONT) and PacBio HiFi sequencing data were generated for the HG00733 lymphoblastoid cell line following specific DNA extraction and purification protocols. Alignment utilized GRCh38 as the reference genome, excluding regions with unreliable variant calls. Public data sets for HG002 and comparison resources from Genome in a Bottle (GIAB) were incorporated, and F1 scores measured variant call accuracy. Generated data were provided to the NCBI BioProject under accession PRJNA966152.
Results of the study showed that PacBio HiFi generally offers better recall, especially for single nucleotide variants (SNVs) and small indels, with optimal performance around 12x coverage. Both methods reveal complementary strengths: ONT excels at detecting large insertions, probably due to longer reads, while HiFi assemblies perform well at deeper coverages. New ONT duplex sequencing improved accuracy and indel recall in low coverage settings, while PacBio Revio systems boosted throughput.
In summary, this study discussed the strengths of LRS platforms and methods for variant discovery. For SNVs, coverage over 12x yields diminishing returns. Both platforms, PacBio HiFi and ONT, have advantages in specific applications: ONT excels in detecting large insertions, while PacBio HiFi shows high recall across all coverages. Deeper coverage (a minimum of 15-20x) is necessary for resolving complex variants and generating high-precision assemblies. Hybrid approaches that integrate PacBio HiFi and ONT data show promise for creating comprehensive telomere-to-telomere (T2T) genome assemblies, potentially advancing variant discovery.
Abbreviations
LRS: Long-Read Sequencing
ONT: Oxford Nanopore Technologies
PacBio: PacBio Biosciences
SV: Structural Variant
HG00733: Specific Lymphoblastoid Cell Line
GRCh38: Genome Reference Consortium Human Build 38
HG002: Specific Human Genome Sample
GIAB: Genome in a Bottle
F1 Score: F1 Score (a measure of variant call accuracy)
NCBI: National Center for Biotechnology Information
PRJNA966152: NCBI BioProject Accession Number
SNV: Single Nucleotide Variant
indel: Insertion/Deletion
T2T: Telomere-to-Telomere
