With the rapid development of sequencing technology and the dramatic decrease of sequencing costs, DNA sequencing has been widely used in scientific research, diagnosis of and treatment selection for human diseases. However, due to the lack of effective quality assessment and control of the high-throughput omics data generation and analysis processes, variants calling results are seriously inconsistent among different technical replicates, batches, laboratories, sequencing platforms, and analysis pipelines, resulting in irreproducible scientific results and conclusions, huge waste of resources, and even endangering the life and health of patients. Therefore, reference materials for quality control of the whole process from omics data generation to data analysis are urgently needed.
We established four immortalized lymphoblastoid cell lines of a Chinese Quartet family, including father (F7), mother (M8), and monozygotic twin daughters (D5 and D6). The Quartet DNA reference materials are genomic DNA (gDNA) extracted from each immortalized lymphoblastoid cell line in large single batches. They have been certified by China’s State Administration for Market Regulation as the First Class of National Reference Materials and are extensively being utilized for proficiency testing and method validation. The certified reference material numbers are GBW09900 (D5), GBW09901 (D6), GBW09902 (F7), and GBW09903 (M8).
To establish small variant benchmark calls, we selected variants concordant in multiple call sets and Mendelian consistency in the Quartet family. First, we kept variants supported by most call sets. Small-variant genotypes were retained, if supported by (1) at least 2/3 replicates in one batch, (2) at least 4/5 batches by PCR library preparation and 3/4 batches by PCR-free, and (3) both PCR and PCR-free library preparation methods. More than 6 million variants were called across 27 gVCF call sets for each Quartet sample, and 1.4 million irreproducible variants were removed by this filtering process.
We then checked Mendelian inheritance status within the Quartet family of father-mother-twins for those remained reproducible variants. The number of Mendelian violations was much higher than what would be expected due to de novo mutations or somatic mutations arose from cell culture, therefore Mendelian violations are assumed to be probably artifacts. We filtered 0.45 million Mendelian violated small variants and kept 4.2 million consensus small variants for each Quartet DNA reference material, which were shared by the identical twins and followed Mendelian inheritance laws with parents.
Small variant benchmark calls were validated by PacBio circular consensus sequencing (CCS) reads and the Axiom Precision Medicine Research Array (PMRA). False positives confirmed by Integrative Genomics Viewer (IGV) were removed.
We used the same strategy to establish structural variant benchmark calls. Since the detection of duplications (DUPs), inversions (INVs) and breakends (BNDs) varied between different pipelines, we only considered insertions (INSs) and deletions (DELs) into structural variant benchmark calls. To select reproducible structural variants, we first merged clustered variants of the same type in a single call set, for a large structural variant may be incorrectly called as multiple adjacent small structural variants. This left ~90,000 structural variants in 30 call sets for each Quartet sample. Reproducible structural variants were selected if supported by at least six pipelines of one platform or two sequencing platforms by any pipeline. Then, large structural variants (over 10Mb) and structural variants located in centromeres, peri-centromere, and gaps regions were excluded.
We merged the four Quartet integrated structural variants catalogs and re-genotyped them in a pedigree by three genotypers (Sniffles, SVjedi, LRcaller) from two sequencing platforms (PacBio Sequel and ONT). A total of 23,891 structural variants were retained, which were supported by at least 6/10 re-genotyped call sets. Among them, 4760 structural variants were Mendelian violations and removed. Finally, about 15,000 structural variants were kept as benchmark calls for each Quartet DNA reference material. We observed expected peaks for INSs and DELs near 300bp, 2.1kb and 6kb corresponding to Alu elements, SVA elements and full-length LINE1 elements respectively, consistent with published studies. Structural variant benchmark calls were then validated by short-read, linked-read and optical and long-read assembly-based call sets.
Benchmark regions of structural variants were established from high-depth sequencing PacBio Sequel assemblies. The benchmark regions of the twin’s cover ~2.62Gb of the reference genome (GRCh38; chr1-22), which contain 12,705 structural variants (7008 INSs and 5697 DELs). The benchmark regions of F7 cover ~2.59 Gb and 12,830 structural variants (6785 INSs and 6045 DELs). The benchmark regions of M8 cover ~2.59Gb and 12,634 structural variants (6568 INSs and 6066 DELs).
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Quartet DNA reference materials
Quartet Data Portal https://chinese-quartet.org/#/materials
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Quartet DNA benchmark sets
Quartet Data Portal https://chinese-quartet.org/#/reference-datasets/download