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Genomic profiling of circulating tumor DNA derived from cell-free DNA (cfDNA) in blood can provide a noninvasive method for detecting genomic biomarkers to guide clinical decision making for cancer patients. We developed a hybrid capture-based next-generation sequencing assay for genomic profiling of circulating tumor DNA from blood (FoundationACT). High-sequencing coverage and molecular barcode-based error detection enabled accurate detection of genomic alterations, including short variants (base substitutions, short insertions/deletions) and genomic re-arrangements at low allele frequencies (AFs), and copy number amplifications. Analytical validation was performed on 2666 reference alterations. The assay achieved >99% overall sensitivity (95% CI, 99.1%-99.4%) for short variants at AF >0.5%, >95% sensitivity (95% CI, 94.2%-95.7%) for AF 0.25% to 0.5%, and 70% sensitivity (95% CI, 68.2%-71.5%) for AF 0.125% to 0.25%. No false positives were detected in 62 samples from healthy volunteers. Genomic alterations detected by FoundationACT demonstrated high concordance with orthogonal assays run on the same clinical cfDNA samples. In 860 routine clinical FoundationACT cases, genomic alterations were detected in cfDNA at comparable frequencies to tissue; for the subset of cases with temporally matched tissue and blood samples, 75% of genomic alterations and 83% of short variant mutations detected in tissue were also detected in cfDNA. On the basis of analytical validation results, FoundationACT has been approved for use in our Clinical Laboratory Improvement Amendments-certified/College of American Pathologists-accredited/New York State-approved laboratory., (Copyright © 2018 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Here we report the Simons Genome Diversity Project data set: high quality genomes from 300 individuals from 142 diverse populations. These genomes include at least 5.8 million base pairs that are not present in the human reference genome. Our analysis reveals key features of the landscape of human genome variation, including that the rate of accumulation of mutations has accelerated by about 5% in non-Africans compared to Africans since divergence. We show that the ancestors of some pairs of present-day human populations were substantially separated by 100,000 years ago, well before the archaeologically attested onset of behavioural modernity. We also demonstrate that indigenous Australians, New Guineans and Andamanese do not derive substantial ancestry from an early dispersal of modern humans; instead, their modern human ancestry is consistent with coming from the same source as that of other non-Africans.

Next generation sequencing is a transformative technology for discovering and diagnosing genetic disorders. However, high-throughput sequencing remains error-prone, necessitating variant confirmation in order to meet the exacting demands of clinical diagnostic sequencing. To address this, we devised an orthogonal, dual platform approach employing complementary target capture and sequencing chemistries to improve speed and accuracy of variant calls at a genomic scale. We combined DNA selection by bait-based hybridization followed by Illumina NextSeq reversible terminator sequencing with DNA selection by amplification followed by Ion Proton semiconductor sequencing. This approach yields genomic scale orthogonal confirmation of ~95% of exome variants. Overall variant sensitivity improves as each method covers thousands of coding exons missed by the other. We conclude that orthogonal NGS offers improvements in variant calling sensitivity when two platforms are used, better specificity for variants identified on both platforms, and greatly reduces the time and expense of Sanger follow-up, thus enabling physicians to act on genomic results more quickly.

Genetic studies have consistently indicated a single common origin of Native American groups from Central and South America. However, some morphological studies have suggested a more complex picture, whereby the northeast Asian affinities of present-day Native Americans contrast with a distinctive morphology seen in some of the earliest American skeletons, which share traits with present-day Australasians (indigenous groups in Australia, Melanesia, and island Southeast Asia). Here we analyse genome-wide data to show that some Amazonian Native Americans descend partly from a Native American founding population that carried ancestry more closely related to indigenous Australians, New Guineans and Andaman Islanders than to any present-day Eurasians or Native Americans. This signature is not present to the same extent, or at all, in present-day Northern and Central Americans or in a ∼12,600-year-old Clovis-associated genome, suggesting a more diverse set of founding populations of the Americas than previously accepted.

Traditional carrier screening assays are designed to look for only the most common mutations within a gene owing to cost considerations. Although this can yield high detection rates in specific populations for specific genes (such as cystic fibrosis in Caucasians), they are suboptimal for other ethnicities or for patients of mixed or unknown ethnic background. Next-generation DNA sequencing provides an opportunity to provide carrier screening using more comprehensive mutation panels that are limited primarily by information about the clinical impact of detected sequence changes. We describe a next-generation DNA sequencing-based assay capable of reliably screening patient samples in a timely and comprehensive manner. The analytic accuracy in a research setting has been documented. Here, we describe the additional studies performed to ensure the accuracy (analytic validity) and robustness of our assay for use in clinical practice and provide data from our experience offering this testing. Our clinical experience using this approach to screen 11,691 in vitro fertilization patients has identified 449 mutant alleles: 447 in carriers and 2 in an affected individual. In total, we found 87 distinct mutations in 14 different genes. Approximately one quarter of the mutations found are not included in traditional, limited, mutation panels, including 16 known mutations unique to our panel, and novel truncating mutations in several genes., (Copyright © 2014 American Society for Investigative Pathology and the Association for Molecular Pathology. Published by Elsevier Inc. All rights reserved.)

Purpose: Carrier screening for recessive Mendelian disorders traditionally employs focused genotyping to interrogate limited sets of mutations most prevalent in specific ethnic groups. We sought to develop a next-generation DNA sequencing-based workflow to enable analysis of a more comprehensive set of disease-causing mutations., Methods: We utilized molecular inversion probes to capture the protein-coding regions of 15 genes from genomic DNA isolated from whole blood and sequenced those regions using the Illumina HiSeq 2000 (Illumina, San Diego, CA). To assess the quality of the resulting data, we measured both the fraction of the targeted region yielding high-quality genotype calls, and the sensitivity and specificity of those calls by comparison with conventional Sanger sequencing across hundreds of samples. Finally, to improve the overall accuracy for detecting insertions and deletions, we introduce a novel assembly-based approach that substantially increases sensitivity without reducing specificity., Results: We generated high-quality sequence for at least 99.8% of targeted base pairs in samples derived from blood and achieved high concordance with Sanger sequencing (sensitivity >99.9%, specificity >99.999%). Our novel algorithm is capable of detecting insertions and deletions inaccessible by current methods., Conclusion: Our next-generation DNA sequencing-based approach yields the accuracy and completeness necessary for a carrier screening test.

Background: Epidemiologic studies require identification or typing of microbial strains. Macrorestriction DNA mapping analyzed by pulsed-field gel electrophoresis (PFGE) is considered the current gold standard of genomic typing. This technique, however, is difficult to implement because it is labor-intensive and difficult to automate, it requires a long time to obtain results, and results often vary between laboratories., Methods: We used direct linear analysis (DLA), which uses a single reagent set and long fragments of microbial genomic DNA to identify various microbes. In this technique, an automated system extracts fragments exceeding 100 kb from restriction enzyme digests of genomic DNA from microbial isolates and hybridizes them with a sequence-dependent fluorescent tag. These fragments are then stretched in a microfluidics chip, and the patterns of the distribution of the tags are discerned with fluorescence confocal microscopy. The tag pattern on each DNA fragment is compared with a database of known microbial DNA sequences or with measured patterns of other microbial DNAs., Results: We used DLA to type 71 Staphylococcus aureus strains. Of these, 9 had been sequenced, 10 were representative of the major pulsed-field types present in the US, and 52 were isolated recently in a hospital in Cambridge, MA. Matching DNA fragments were identified in different samples by a clustering algorithm and were used to quantify the similarities of the strains., Conclusions: DLA-based strain typing is a powerful technique with a resolution comparable to macrorestriction mapping with PFGE, but DLA is faster, more automated, and more reproducible.