We report the sequencing and assembly of a reference genome for the human GM12878 Utah/Ceph cell line using the MinION (Oxford Nanopore Technologies) nanopore sequencer. 91.2 Gb of sequence data, representing ~30× theoretical coverage, were produced. Reference-based alignment enabled detection of large structural variants and epigenetic modifications. De novo assembly of nanopore reads alone yielded a contiguous assembly (NG50 ~3 Mb). Next, we developed a protocol to generate ultra-long reads (N50 > 100kb, up to 882 kb). Incorporating an additional 5×-coverage of this data type more than doubled the assembly contiguity (NG50 ~6.4 Mb). The final assembled genome was 2,867 million bases in size, covering 85.8% of the reference. Assembly accuracy, after incorporating complementary short-read sequencing data, exceeded 99.8%. Ultra-long reads enabled assembly and phasing of the 4 Mb major histocompatibility complex (MHC) locus in its entirety, measurement of telomere repeat length and closure of gaps in the reference human genome assembly GRCh38.

Abbreviations: CAD = Coronary artery disease; SNPs = Single-nucleotide polymorphisms; E2 = 17β-estradiol; T = Testosterone; A = Aromatase ABSTRACT Background: The relationship between CYP19A1 genetic polymorphisms and coronary artery disease (CAD) remains unclear. Thus, the aim of the present study was to investigate the association of CYP19A1 genetic polymorphisms with CAD in Han and Uygur populations and to characterize the association between the levels of sex hormones and aromatase with single-nucleotide polymorphisms (SNPs) in CYP19A1 genes in Chinese women. Results: There were significant differences in the genotype distributions of rs2236722 and rs4646 between CAD patients and control subjects in the Uygur population. The rs4646 was found to be associated with CAD in the dominant model (CC vs. CA + AA) and the additive model (CA vs. CC + AA) (both P ≤ 0.001). The difference remained statistically significant after multivariate adjustment (OR = 0.483, 95% CI: 0.338-0.690, P = 0.000; and OR = 1.844, 95% CI: 1.300-2.617, P = 0.001, respectively). In normal Uygur postmenopausal women, there were significant differences in the genotype distributions of rs4646 and the circulating hormone and aromatase levels between CAD patients and control subjects. The differences in estradiol and aromatase levels remained statistically significant after multivariate adjustment (OR = 0.889, 95% CI: 0.817-0.969, P = 0.007; and OR = 0.947, 95% CI: 0.936-0.957, P = 0.000, respectively). Additionally, there were differences in sex hormone levels between the different ethnicities among the Xinjiang Chinese population. Materials and Methods: Among a total of 1,064 Han individuals (614 men and 450 women) and 790 Uygur individuals (484 men and 306 women), 498 postmenopausal women (265 Han and 233 Uygur individuals) were selected. Four SNPs (rs2236722, rs2304463, rs4646, and rs4275794) were genotyped using the improved multiplex ligation detection reaction (iMLDR) technique. The estradiol and testosterone levels were determined using a radioimmunoassay based on GC-2016γ. In addition, an enzyme-linked immunosorbent assay (ELISA) was performed to determine the serum P450 aromatase levels. Conclusions: The results of this study indicate that the rs2236722 and rs4646 of the CYP19A1 gene are associated with CAD and circulating sex hormone levels in the Xinjiang population of China.

Meckel-Gruber syndrome is a severe autosomal, recessively inherited disorder characterized by bilateral renal cystic dysplasia, developmental defects of the central nervous system (most commonly occipital encephalocele), hepatic ductal dysplasia and cysts and polydactyly. MKS is genetically heterogeneous, with three loci mapped: MKS1, 17q21-24 (ref. 4); MKS2, 11q13 (ref. 5) and MKS3 (ref. 6). We have refined MKS3 mapping to a 12.67-Mb interval (8q21.13-q22.1) that is syntenic to the Wpk locus in rat, which is a model with polycystic kidney disease, agenesis of the corpus callosum and hydrocephalus. Positional cloning of the Wpk gene suggested a MKS3 candidate gene, TMEM67, for which we identified pathogenic mutations for five MKS3-linked consanguineous families. MKS3 is a previously uncharacterized, evolutionarily conserved gene that is expressed at moderate levels in fetal brain, liver and kidney but has widespread, low levels of expression. It encodes a 995-amino acid seven-transmembrane receptor protein of unknown function that we have called meckelin.

Examining complete gene knockouts within a viable organism can inform on gene function. We sequenced the exomes of 3,222 British Pakistani-heritage adults with high parental relatedness, discovering 1,111 rare-variant homozygous genotypes with predicted loss of gene function (knockouts) in 781 genes. We observed 13.7% fewer than expected homozygous knockout genotypes, implying an average load of 1.6 recessive-lethal-equivalent LOF variants per adult. Linking genetic data to lifelong health records, knockouts were not associated with clinical consultation or prescription rate. In this dataset we identified a healthy PRDM9 knockout mother, and performed phased genome sequencing on her, her child and controls, which showed meiotic recombination sites localised away from PRDM9-dependent hotspots. Thus, natural LOF variants inform upon essential genetic loci, and demonstrate PRDM9 redundancy in humans.

Genetic variants of the SLC6A3 gene that encodes the human dopamine transporter (DAT) have been linked to a variety of neuropsychiatric disorders, particularly attention deficit hyperactivity disorder. In addition, the homozygous Slc6a3 knockout mouse displays a hyperactivity phenotype. Here, we analyzed 2 unrelated consanguineous families with infantile parkinsonism-dystonia (IPD) syndrome and identified homozygous missense SLC6A3 mutations (p.L368Q and p.P395L) in both families. Functional studies demonstrated that both mutations were loss-of-function mutations that severely reduced levels of mature (85-kDa) DAT while having a differential effect on the apparent binding affinity of dopamine. Thus, in humans, loss-of-function SLC6A3 mutations that impair DAT-mediated dopamine transport activity are associated with an early-onset complex movement disorder. Identification of the molecular basis of IPD suggests SLC6A3 as a candidate susceptibility gene for other movement disorders associated with parkinsonism and/or dystonic features.