Genomic Analysis in the Age of Human Genome Sequencing.

Affordable genome sequencing technologies promise to revolutionize the field of human genetics by enabling comprehensive studies that interrogate all classes of genome variation, genome-wide, across the entire allele frequency spectrum. Ongoing projects worldwide are sequencing many thousands-and soon millions-of human genomes as part of various gene mapping studies, biobanking efforts, and clinical programs. However, while genome sequencing data production has become routine, genome analysis and interpretation remain challenging endeavors with many limitations and caveats. Here, we review the current state of technologies for genetic variant discovery, genotyping, and functional interpretation and discuss the prospects for future advances. We focus on germline variants discovered by whole-genome sequencing, genome-wide functional genomic approaches for predicting and measuring variant functional effects, and implications for studies of common and rare human disease.

Toward the single-hour high-quality genome.

Today, resequencing of a human genome can be performed in approximately a week using a single instrument. Thanks to a steady logarithmic rate of increase in performance for DNA sequencing platforms over the past seven years, DNA sequencing is one of the fastest developing technology fields. As the process becomes faster, it opens up possibilities within health care, diagnostics, and entirely new fields of research. Immediate genetic characterization of contagious outbreaks has been exemplified, and with such applications for the direct benefit of human health, expectations of future sensitive, rapid, high-throughput, and cost-effective technologies are steadily growing. Simultaneously, some of the limitations of a rapidly growing field have become apparent, and questions regarding the quality of some of the data deposited into databases have been raised. A human genome sequenced in only an hour is likely to become a reality in the future, but its definition may not be as certain.

Exome sequencing deciphers rare diseases.

Two years ago, NIH's Undiagnosed Diseases Program began delivering genomics to the clinic on an unprecedented scale. Now, with 128 exomes sequenced and 39 rare diseases diagnosed, the program's success is paving the way for widespread personal genomics while pioneering new techniques for reigning in the "tsunami" of genomics data.

Whole-genome sequencing breaks the cost barrier.

The ever-decreasing cost of sequencing is ushering in a new era in genomic medicine. Laura Bonetta reports on new studies that provide a glimpse of the opportunities ahead.

DNA sequencing at 40: past, present and future.

This review commemorates the 40th anniversary of DNA sequencing, a period in which we have already witnessed multiple technological revolutions and a growth in scale from a few kilobases to the first human genome, and now to millions of human and a myriad of other genomes. DNA sequencing has been extensively and creatively repurposed, including as a 'counter' for a vast range of molecular phenomena. We predict that in the long view of history, the impact of DNA sequencing will be on a par with that of the microscope.

Cell-free fetal DNA coming in all sizes and shapes.

Cell-free fetal DNA analysis has an established role in prenatal assessments. It serves as a source of fetal genetic material that is accessible non-invasively from maternal blood. Through the years, evidence has accumulated to show that cell-free fetal DNA molecules are derived from placental tissues, are mainly of short DNA fragments and have rapid post-delivery clearance profiles. But questions regarding how they come to being short molecules from placental cells and in which physical forms do they exist remained largely unanswered until recently. We now know that the distributions of ending sites of cell-free DNA molecules are non-random across the genome and bear correlations with the chromatin structures of cells from which they have originated. Such an insight offers ways to deduce the tissue-of-origin of these molecules. Besides, the physical nature and sequence characteristics of the ends of each cell-free DNA molecule provide tell-tale signs of how the DNA fragmentation processes are orchestrated by nuclease enzymes. These realizations offered opportunities to develop methods for enriching cell-free fetal DNA to facilitate non-invasive prenatal diagnostics. Here we aimed to collate what is known about the biological and physical characteristics of cell-free fetal DNA into one article and explain the implications of these observations.

Genomic Tackling of Human Satellite DNA: Breaking Barriers through Time.

(Peri)centromeric repetitive sequences and, more specifically, satellite DNA (satDNA) sequences, constitute a major human genomic component. SatDNA sequences can vary on a large number of features, including nucleotide composition, complexity, and abundance. Several satDNA families have been identified and characterized in the human genome through time, albeit at different speeds. Human satDNA families present a high degree of sub-variability, leading to the definition of various subfamilies with different organization and clustered localization. Evolution of satDNA analysis has enabled the progressive characterization of satDNA features. Despite recent advances in the sequencing of centromeric arrays, comprehensive genomic studies to assess their variability are still required to provide accurate and proportional representation of satDNA (peri)centromeric/acrocentric short arm sequences. Approaches combining multiple techniques have been successfully applied and seem to be the path to follow for generating integrated knowledge in the promising field of human satDNA biology.

Applying high-throughput sequencing to identify and evaluate foetal chromosomal deletion and duplication.

The present study aimed to estimate the clinical performance of non-invasive prenatal testing (NIPT) based on high-throughput sequencing method for the detection of foetal chromosomal deletions and duplications. A total of 6348 pregnant women receiving NIPT using high-throughput sequencing method were included in our study. They all conceived naturally, without twins, triplets or multiple births. Individuals showing abnormalities in NIPT received invasive ultrasound-guided amniocentesis for chromosomal karyotype and microarray analysis at 18-24 weeks of pregnancy. Detection results of foetal chromosomal deletions and duplications were compared between high-throughput sequencing method and chromosomal karyotype and microarray analysis. Thirty-eight individuals were identified to show 51 chromosomal deletions/duplications via high-throughput sequencing method. In subsequent chromosomal karyotype and microarray analysis, 34 subchromosomal deletions/duplications were identified in 26 pregnant women. The observed deletions and duplications ranged from 1.05 to 17.98 Mb. Detection accuracy for these deletions and duplications was 66.7%. Twenty-one deletions and duplications were found to be correlated with the known abnormalities. NIPT based on high-throughput sequencing technique is able to identify foetal chromosomal deletions and duplications, but its sensitivity and specificity were not explored. Further progress should be made to reduce false-positive results.

Massive parallel sequencing in forensics: advantages, issues, technicalities, and prospects.

In the last decade, next-generation sequencing (NGS) technology, alternatively massive parallel sequencing (MPS), was applied to all fields of biological research. Its introduction to the field of forensics was slower, mainly due to lack of accredited sequencers, kits, and relatively higher sequencing error rates as compared with standardized Sanger sequencing. Currently, a majority of the problematic issues have been solved, which is proven by the body of reports in the literature. Here, we discuss the utility of NGS sequencing in forensics, emphasizing the advantages, issues, the technical aspects of the experiments, commercial solutions, and the potentially interesting applications of MPS.

Tools for Analysis of the Microbiome.

Over the past decade, it has become exceedingly clear that the microbiome is a critical factor in human health and disease and thus should be investigated to develop innovative treatment strategies. The field of metagenomics has come a long way in leveraging the advances of next-generation sequencing technologies resulting in the capability to identify and quantify all microorganisms present in human specimens. However, the field of metagenomics is still in its infancy, specifically in regard to the limitations in computational analysis, statistical assessments, standardization, and validation due to vast variability in the cohorts themselves, experimental design, and bioinformatic workflows. This review summarizes the methods, technologies, computational tools, and model systems for characterizing and studying the microbiome. We also discuss important considerations investigators must make when interrogating the involvement of the microbiome in health and disease in order to establish robust results and mechanistic insights before moving into therapeutic design and intervention.

Race and Genetics: Somber History, Troubled Present.

Following the completion of the Human Genome Project (HGP) in 2003, advances in DNA sequencing technologies further popularized the field of genomics and brought its social ramifications to the fore. Scholars across disciplines recently voiced serious concerns about the re-emergence of genomic research that might be used to justify racism. In this piece, I trace the history of attempts to biologize the concept of race and its diffused presence in today's genomic research. I then include a brief analysis inspired by concepts from the field of Science and Technology Studies (STS) to suggest selected ways to produce better scientific knowledge. The text highlights historic landmarks of interest to science practitioners curious about the ways science of the past co-shapes science of the present. I then argue that science has never been isolated from the socio-political climate it is produced in; instead, it has been morphed by its surroundings and historically used as a potent tool to justify systemic oppression.

Pharma-Oncogenomics in the Era of Personal Genomics: A Quick Guide to Online Resources and Tools.

The past two decades have seen unprecedented advances in the field of oncogenomics. The ongoing characterization of neoplastic tissues through genomic techniques has transformed many aspects of cancer research, diagnosis, and treatment. However, identifying sequence variants with biological and clinical significance is a challenging endeavor. In order to accomplish this task, variants must be annotated and interpreted using various online resources. Data on protein structure, functional prediction, variant frequency in relevant populations, and multiple other factors have been compiled in useful databases for this purpose. Thus, understanding the available online resources for the annotation and interpretation of sequence variants is critical to aid molecular pathologists and researchers working in this space.

Protein Science by DNA Sequencing: How Advances in Molecular Biology Are Accelerating Biochemistry.

A fundamental goal of protein biochemistry is to determine the sequence-function relationship, but the vastness of sequence space makes comprehensive evaluation of this landscape difficult. However, advances in DNA synthesis and sequencing now allow researchers to assess the functional impact of every single mutation in many proteins, but challenges remain in library construction and the development of general assays applicable to a diverse range of protein functions. This Perspective briefly outlines the technical innovations in DNA manipulation that allow massively parallel protein biochemistry and then summarizes the methods currently available for library construction and the functional assays of protein variants. Areas in need of future innovation are highlighted with a particular focus on assay development and the use of computational analysis with machine learning to effectively traverse the sequence-function landscape. Finally, applications in the fundamentals of protein biochemistry, disease prediction, and protein engineering are presented.

Genome sequencing in the clinic: the past, present, and future of genomic medicine.

Genomic sequencing has undergone massive expansion in the past 10 yr, from a rarely used research tool into an approach that has broad applications in a clinical setting. From rare disease to cancer, genomics is transforming our knowledge of biology. The transition from targeted gene sequencing, to whole exome sequencing, to whole genome sequencing has only been made possible due to rapid advancements in technologies and informatics that have plummeted the cost per base of DNA sequencing and analysis. The tools of genomics have resolved the etiology of disease for previously undiagnosable conditions, identified cancer driver gene variants, and have impacted the understanding of pathophysiology for many diseases. However, this expansion of use has also highlighted research's current voids in knowledge. The lack of precise animal models for gene-to-function association, lack of tools for analysis of genomic structural changes, skew in populations used for genetic studies, publication biases, and the "Unknown Proteome" all contribute to voids needing filled for genomics to work in a fast-paced clinical setting. The future will hold the tools to fill in these voids, with new data sets and the continual development of new technologies allowing for expansion of genomic medicine, ushering in the days to come for precision medicine. In this review we highlight these and other points in hopes of advancing and guiding precision medicine into the future for optimal success.

Next-generation sequencing data interpretation: enhancing reproducibility and accessibility.

Areas of life sciences research that were previously distant from each other in ideology, analysis practices and toolkits, such as microbial ecology and personalized medicine, have all embraced techniques that rely on next-generation sequencing instruments. Yet the capacity to generate the data greatly outpaces our ability to analyse it. Existing sequencing technologies are more mature and accessible than the methodologies that are available for individual researchers to move, store, analyse and present data in a fashion that is transparent and reproducible. Here we discuss currently pressing issues with analysis, interpretation, reproducibility and accessibility of these data, and we present promising solutions and venture into potential future developments.