Genome sequencing of large numbers of individuals promises to advance the understanding, treatment, and prevention of human diseases, among other applications. We describe a genome sequencing platform that achieves efficient imaging and low reagent consumption with combinatorial probe anchor ligation chemistry to independently assay each base from patterned nanoarrays of self-assembling DNA nanoballs. We sequenced three human genomes with this platform, generating an average of 45- to 87-fold coverage per genome and identifying 3.2 to 4.5 million sequence variants per genome. Validation of one genome data set demonstrates a sequence accuracy of about 1 false variant per 100 kilobases. The high accuracy, affordable cost of $4400 for sequencing consumables, and scalability of this platform enable complete human genome sequencing for the detection of rare variants in large-scale genetic studies.

ObjectiveTo develop a novel prenatal assay based on selective analysis of cell-free DNA in maternal blood for evaluation of fetal Trisomy 21 (T21) and Trisomy 18 (T18).MethodsTwo hundred ninety-eight pregnancies, including 39 T21 and seven T18 confirmed fetal aneuploidies, were analyzed using a novel, highly multiplexed assay, termed digital analysis of selected regions (DANSR™). Cell-free DNA from maternal blood samples was analyzed using DANSR assays for loci on chromosomes 21 and 18. Products from 96 separate patients were pooled and sequenced together. A standard Z-test of chromosomal proportions was used to distinguish aneuploid samples from average-risk pregnancy samples. DANSR aneuploidy discrimination was evaluated at various sequence depths.ResultsAt the lowest sequencing depth, corresponding to 204 000 sequencing counts per sample, average-risk cases where distinguished from T21 and T18 cases, with Z statistics for all cases exceeding 3.6. Increasing the sequencing depth to 410 000 counts per sample substantially improved separation of aneuploid and average-risk cases. A further increase to 620 000 counts per sample resulted in only marginal improvement. This depth of sequencing represents less than 5% of that required by massively parallel shotgun sequencing approaches.ConclusionDigital analysis of selected regions enables highly accurate, cost efficient, and scalable noninvasive fetal aneuploidy assessment. © 2012 John Wiley & Sons, Ltd.

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NK cell activation is controlled by the integration of signals from cytokine receptors and germ-line encoded activation and inhibitory receptors. NK cells undergo two distinct phases of activation during MCMV infection: a nonselective phase mediated by pro-inflammatory cytokines and a specific phase driven by signaling through Ly49H, an NK cell activation receptor that recognizes infected cells. We sought to delineate cell surface markers that could distinguish NK cells that had been activated nonselectively from those that had been specifically activated through NK cell receptors. We demonstrated that Sca-1 is highly upregulated during viral infections (to an even greater extent than CD69) and serves as a novel marker of early, nonselective NK cell activation. Indeed, a greater proportion of Sca-1+ NK cells produced IFN-γ compared to Sca-1− NK cells during MCMV infection. In contrast to the universal upregulation of Sca-1 (as well as KLRG1) on NK cells early during MCMV infection, differential expression of Sca-1, as well as CD27 and KLRG1, was observed on Ly49H+ and Ly49H− NK cells late during MCMV infection. Persistently elevated levels of KLRG1 in the context of down regulation of Sca-1 and CD27 were observed on NK cells that expressed Ly49H. Furthermore, the differential expression patterns of these cell surface markers were dependent on Ly49H recognition of its ligand and did not occur solely as a result of cellular proliferation. These findings demonstrate that a combination of Sca-1, CD27, and KLRG1 can distinguish NK cells nonselectively activated by cytokines from those specifically stimulated through activation receptors.

Purpose: To investigate the link between treatment with CTLA-4 and PD-1 checkpoint blockade inhibitors and development of noninfectious uveitis. Methods:A survey was distributed to uveitis specialists to identify patients who developed uveitis while receiving either PD-1 inhibitors pembrolizumab and nivolumab; PD-L1 inhibitors atezolizumab, avelumab, and durvalumab; or the CTLA-4 inhibitor ipilimumab.Results: Fifteen patients from seven institutions were identified. The most common cancer diagnosis (13/15) was malignant melanoma. Fourteen patients had a new uveitis diagnosis following checkpoint blockade administration (6 anterior uveitis, 6 panuveitis, 1 posterior uveitis, 1 anterior/intermediate combined); one patient developed optic neuritis. Uveitis was diagnosed within 6 months after drug initiation for 11/12 patients (median 63 days). Corticosteroid treatment was effective for most patients, although 2 patients had permanent loss of vision. Conclusions:Patients on checkpoint inhibitor therapy should be educated to seek care if they develop ocular symptoms, and prompt referral to specialists should be incorporated into oncology protocols.