We report the sequences of 1,244 human Y chromosomes randomly ascertained from 26 worldwide populations by the 1000 Genomes Project. We discovered more than 65,000 variants, including ...single-nucleotide variants, multiple-nucleotide variants, insertions and deletions, short tandem repeats, and copy number variants. Of these, copy number variants contribute the greatest predicted functional impact. We constructed a calibrated phylogenetic tree on the basis of binary single-nucleotide variants and projected the more complex variants onto it, estimating the number of mutations for each class. Our phylogeny shows bursts of extreme expansion in male numbers that have occurred independently among each of the five continental superpopulations examined, at times of known migrations and technological innovations.
The Long interspersed nuclear element 1 (LINE-1) is a primary source of genetic variation in humans and other mammals. Despite its importance, LINE-1 activity remains difficult to study because of ...its highly repetitive nature. Here, we developed and validated a method called TeXP to gauge LINE-1 activity accurately. TeXP builds mappability signatures from LINE-1 subfamilies to deconvolve the effect of pervasive transcription from autonomous LINE-1 activity. In particular, it apportions the multiple reads aligned to the many LINE-1 instances in the genome into these two categories. Using our method, we evaluated well-established cell lines, cell-line compartments and healthy tissues and found that the vast majority (91.7%) of transcriptome reads overlapping LINE-1 derive from pervasive transcription. We validated TeXP by independently estimating the levels of LINE-1 autonomous transcription using ddPCR, finding high concordance. Next, we applied our method to comprehensively measure LINE-1 activity across healthy somatic cells, while backing out the effect of pervasive transcription. Unexpectedly, we found that LINE-1 activity is present in many normal somatic cells. This finding contrasts with earlier studies showing that LINE-1 has limited activity in healthy somatic tissues, except for neuroprogenitor cells. Interestingly, we found that the amount of LINE-1 activity was associated with the with the amount of cell turnover, with tissues with low cell turnover rates (e.g. the adult central nervous system) showing lower LINE-1 activity. Altogether, our results show how accounting for pervasive transcription is critical to accurately quantify the activity of highly repetitive regions of the human genome.
Comprehensive and accurate identification of structural variations (SVs) from next generation sequencing data remains a major challenge. We develop FusorSV, which uses a data mining approach to ...assess performance and merge callsets from an ensemble of SV-calling algorithms. It includes a fusion model built using analysis of 27 deep-coverage human genomes from the 1000 Genomes Project. We identify 843 novel SV calls that were not reported by the 1000 Genomes Project for these 27 samples. Experimental validation of a subset of these calls yields a validation rate of 86.7%. FusorSV is available at https://github.com/TheJacksonLaboratory/SVE .
Recent studies have demonstrated genetic differences between monozygotic (MZ) twins. To test the hypothesis that early post-twinning mutational events associate with phenotypic discordance, we ...investigated a cohort of 13 twin pairs (n = 26) discordant for various clinical phenotypes using whole-exome sequencing and screened for copy number variation (CNV). We identified a de novo variant in PLCB1, a gene involved in the hydrolysis of lipid phosphorus in milk from dairy cows, associated with lactase non-persistence, and a variant in the mitochondrial complex I gene MT-ND5 associated with amyotrophic lateral sclerosis (ALS). We also found somatic variants in multiple genes (TMEM225B, KBTBD3, TUBGCP4, TFIP11) in another MZ twin pair discordant for ALS. Based on the assumption that discordance between twins could be explained by a common variant with variable penetrance or expressivity, we screened the twin samples for known pathogenic variants that are shared and identified a rare deletion overlapping ARHGAP11B, in the twin pair manifesting with either schizotypal personality disorder or schizophrenia. Parent-offspring trio analysis was implemented for two twin pairs to assess potential association of variants of parental origin with susceptibility to disease. We identified a de novo variant in RASD2 shared by 8-year-old male twins with a suspected diagnosis of autism spectrum disorder (ASD) manifesting as different traits. A de novo CNV duplication was also identified in these twins overlapping CD38, a gene previously implicated in ASD. In twins discordant for Tourette's syndrome, a paternally inherited stop loss variant was detected in AADAC, a known candidate gene for the disorder.
We aimed to develop a whole-genome sequencing (WGS)-based copy number variant (CNV) calling algorithm with the potential of replacing chromosomal microarray assay (CMA) for clinical diagnosis. ...JAX-CNV is thus developed for CNV detection from WGS data. The performance of this CNV calling algorithm was evaluated in a blinded manner on 31 samples and compared to the 112 CNVs reported by clinically validated CMAs for these 31 samples. The result showed that JAX-CNV recalled 100% of these CNVs. Besides, JAX-CNV identified an average of 30 CNVs per individual, respresenting an approximately seven-fold increase compared to calls of clinically validated CMAs. Experimental validation of 24 randomly selected CNVs showed one false positive, i.e., a false discovery rate (FDR) of 4.17%. A robustness test on lower-coverage data revealed a 100% sensitivity for CNVs larger than 300 kb (the current threshold for College of American Pathologists) down to 10× coverage. For CNVs larger than 50 kb, sensitivities were 100% for coverages deeper than 20×, 97% for 15×, and 95% for 10×. We developed a WGS-based CNV pipeline, including this newly developed CNV caller JAX-CNV, and found it capable of detecting CMA-reported CNVs at a sensitivity of 100% with about a FDR of 4%. We propose that JAX-CNV could be further examined in a multi-institutional study to justify the transition of first-tier genetic testing from CMAs to WGS. JAX-CNV is available at https://github.com/TheJacksonLaboratory/JAX-CNV.
Objective
Maternal plasma cell‐free DNA (cfDNA) analysis is a powerful screening tool for Down syndrome. In a pilot series, we examined biologic causes of discordance between the cfDNA test results ...and the fetal karyotype. We also explored the feasibility of obtaining trio biospecimens by using parental engagement.
Methods
A convenience sample of women with discordant cfDNA results were recruited by their care providers. We provided shipping materials and instructions for biospecimen collection. Maternal, newborn, and placental samples were examined with droplet digital PCR.
Results
Thirteen of 15 women successfully had biospecimens obtained remotely. High‐quality DNA was extracted in 12 of 13 women. Presumed biologic etiologies for discordance were identified in 7 of 12 women: 3 cases from additional clinical review (male renal transplant, vanishing twin, and colon cancer) and 4 cases from additional laboratory investigation using droplet digital PCR (3 with confined placental mosaicism and 1 with true fetal mosaicism).
Conclusions
Understanding the biology behind cfDNA‐fetal karyotype discordancy is useful for follow‐up clinical care. Our study suggests that most cases could be resolved by using a trio biospecimen protocol and parental involvement. To improve accuracy, additional sequencing of biospecimens will be required.
What's already known about this topic?
Up to 10% of maternal plasma cell‐free (cf) DNA results are discordant with the fetal or neonatal karyotype. Minimal scientific evidence is available to guide follow‐up management. This has led to inconsistent clinical care.
What does this study add?
Presumed biologic etiologies for discordant cfDNA can be identified in over half of the cases by an in‐depth review of clinical records and/or multispecimen genomic examination. Patients and providers are motivated to participate and will be instrumental in expanding assessments to a larger, more diverse obstetric population.
Structural variants are implicated in numerous diseases and make up the majority of varying nucleotides among human genomes. Here we describe an integrated set of eight structural variant classes ...comprising both balanced and unbalanced variants, which we constructed using short-read DNA sequencing data and statistically phased onto haplotype blocks in 26 human populations. Analysing this set, we identify numerous gene-intersecting structural variants exhibiting population stratification and describe naturally occurring homozygous gene knockouts that suggest the dispensability of a variety of human genes. We demonstrate that structural variants are enriched on haplotypes identified by genome-wide association studies and exhibit enrichment for expression quantitative trait loci. Additionally, we uncover appreciable levels of structural variant complexity at different scales, including genic loci subject to clusters of repeated rearrangement and complex structural variants with multiple breakpoints likely to have formed through individual mutational events. Our catalogue will enhance future studies into structural variant demography, functional impact and disease association.
Short tandem repeats (STRs) and variable number tandem repeats (VNTRs) are important sources of natural and disease-causing variation, yet they have been problematic to resolve in reference genomes ...and genotype with short-read technology. We created a framework tomodel the evolution and instability of STRs and VNTRs in apes. We phased and assembled 3 ape genomes (chimpanzee, gorilla, and orangutan) using long-read and 10x Genomics linkedread sequence data for 21,442 human tandem repeats discovered in 6 haplotype-resolved assemblies of Yoruban, Chinese, and Puerto Rican origin. We define a set of 1,584 STRs/VNTRs expanded specifically in humans, including large tandem repeats affecting coding and noncoding portions of genes (e.g., MUC3A, CACNA1C). We show that short interspersed nuclear element–VNTR–Alu (SVA) retrotransposition is the main mechanism for distributing GC-rich human-specific tandem repeat expansions throughout the genome but with a bias against genes. In contrast, we observe that VNTRs not originating from retrotransposons have a propensity to cluster near genes, especially in the subtelomere. Using tissuespecific expression from human and chimpanzee brains, we identify genes where transcript isoform usage differs significantly, likely caused by cryptic splicing variation within VNTRs. Using single-cell expression from cerebral organoids, we observe a strong effect for genes associated with transcription profiles analogous to intermediate progenitor cells. Finally, we compare the sequence composition of some of the largest human-specific repeat expansions and identify 52 STRs/VNTRs with at least 40 uninterrupted pure tracts as candidates for genetically unstable regions associated with disease.
Copy number variations (CNVs) in the genomes have been suggested to play important roles in human evolution, genetic diversity, and disease susceptibility. A number of assays have been developed for ...the detection of CNVs, including fluorescent in situ hybridization (FISH), array-based comparative genomic hybridization (aCGH), PCR-based assays, and next-generation sequencing (NGS). In this chapter, we describe a microarray method that has been used for the detection of genome-wide CNVs, loss of heterozygosity (LOH), and uniparental disomy (UPD) associated with constitutional and neoplastic disorders.
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