Haplotype-resolved assemblies and variant benchmark of a Chinese Quartet

Abstract

As the state-of-the-art sequencing technologies and computational methods enable investigation of challenging regions in the human genome, an update variant benchmark is demanded. Herein, we sequenced a Chinese Quartet, consisting of two monozygotic twin daughters and their biological parents, with multiple advanced sequencing platforms, including Illumina, BGI, PacBio, and Oxford Nanopore Technology. We phased the long reads of the monozygotic twin daughters into paternal and maternal haplotypes using the parent-child genetic map. For each haplotype, we utilized advanced long reads to generate haplotype-resolved assemblies (HRAs) with high accuracy, completeness, and continuity. Based on the ingenious quartet samples, novel computational methods, high-quality sequencing reads, and HRAs, we established a comprehensive variant benchmark, including 3,883,283 SNVs, 859,256 Indels, 9,678 large deletions, 15,324 large insertions, 40 inversions, and 31 complex structural variants shared between the monozygotic twin daughters. In particular, the preciously excluded regions, such as repeat regions and the human leukocyte antigen (HLA) region, were systematically examined. Finally, we illustrated how the sequencing depth correlated with the de novo assembly and variant detection, from which we learned that 30 X HiFi is a balance between performance and cost. In summary, this study provides high-quality haplotype-resolved assemblies and a variant benchmark for two Chinese monozygotic twin samples. The benchmark expanded the regions of the previous report and adapted to the evolving sequencing technologies and computational methods.

Multi-omics data integration using ratio-based quantitative profiling with Quartet reference materials

“…Four accompanying papers detail the establishment of the DNA 66 , RNA 67 , protein 68 and metabolite 69 reference materials, reference datasets and QC methods for each type of omics profiling. Haplotype-resolved assemblies and a variant benchmark have also been provided 70 . Another paper 71 is dedicated to benchmarking batch effect correction algorithms (BECAs) using the Quartet multi-omics data.…”

Quartet DNA reference materials and datasets for comprehensively evaluating germline variants calling performance

“…BioNano only validated 3.2% INS and 1.8% DEL over 1 kb, due to its low resolution (kb) by specific restriction enzyme cut sites and failure to accurately determine breakpoints 37 . We also validated our SV benchmark callset with Jia et al 34 , and found that 97.1% INS and 91.9% DEL were confirmed.…”

“…We further compared the small variants benchmark calls with high confidence call sets from two accompanying studies 33, 34 (Supplementary Fig. 4).…”

Toward best practices for detecting germline small variants: a large-scale real-world WES benchmarking study using the Quartet DNA reference materials

“…Furthermore, existing benchmark studies typically rely on a single gDNA reference sample and its corresponding benchmark variants, limiting their assessment solely within prede ned high-con dence regions or benchmark regions, while disregarding variants beyond these boundaries. Recently, the Quartet project, aimed for quality control and data integration of multiomic pro ling, established a suite of DNA reference materials from four immortalized cell lines derived from a family of parents and monozygotic twins [21][22][23] . These Quartet DNA reference materials not only facilitate the evaluation of variant calling accuracy within high-con dence regions but also provide pedigree information from the Quartet family design and the Mendelian consistent rate (MCR), thereby extending performance assessment capabilities outside the high-con dence regions to evaluate the precision of variant calls 22 .…”