The human genome holds an extraordinary trove of information about human development, physiology, medicine and evolution. Here we report the results of an international collaboration to produce and make freely available a draft sequence of the human genome. We also present an initial analysis of the data, describing some of the insights that can be gleaned from the sequence.

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Initial sequencing and analysis of the human genome.

Next-generation DNA sequencing (NGS) projects, such as the 1000 Genomes Project, are already revolutionizing our understanding of genetic variation among individuals. However, the massive data sets generated by NGS—the 1000 Genome pilot alone includes nearly five terabases—make writing feature-rich, efficient, and robust analysis tools difficult for even computationally sophisticated individuals. Indeed, many professionals are limited in the scope and the ease with which they can answer scientific questions by the complexity of accessing and manipulating the data produced by these machines. Here, we discuss our Genome Analysis Toolkit (GATK), a structured programming framework designed to ease the development of efficient and robust analysis tools for next-generation DNA sequencers using the functional programming philosophy of MapReduce. The GATK provides a small but rich set of data access patterns that encompass the majority of analysis tool needs. Separating specific analysis calculations from common data management infrastructure enables us to optimize the GATK framework for correctness, stability, and CPU and memory efficiency and to enable distributed and shared memory parallelization. We highlight the capabilities of the GATK by describing the implementation and application of robust, scale-tolerant tools like coverage calculators and single nucleotide polymorphism (SNP) calling. We conclude that the GATK programming framework enables developers and analysts to quickly and easily write efficient and robust NGS tools, many of which have already been incorporated into large-scale sequencing projects like the 1000 Genomes Project and The Cancer Genome Atlas.

/pdf/the-genome-analysis-toolkit-a-mapreduce-framework-for-4aygfa9b66.pdf

The Genome Analysis Toolkit: A MapReduce framework for analyzing next-generation DNA sequencing data

Pfam is a widely used database of protein families and domains. This article describes a set of major updates that we have implemented in the latest release (version 24.0). The most important change is that we now use HMMER3, the latest version of the popular profile hidden Markov model package. This software is approximately 100 times faster than HMMER2 and is more sensitive due to the routine use of the forward algorithm. The move to HMMER3 has necessitated numerous changes to Pfam that are described in detail. Pfam release 24.0 contains 11,912 families, of which a large number have been significantly updated during the past two years. Pfam is available via servers in the UK (http://pfam.sanger.ac.uk/), the USA (http://pfam.janelia.org/) and Sweden (http://pfam.sbc.su.se/).

/pdf/the-pfam-protein-families-database-3t3lguk949.pdf

The Pfam protein families database

Sequence similarity searching is a very important bioinformatics task. While Basic Local Alignment Search Tool (BLAST) outperforms exact methods through its use of heuristics, the speed of the current BLAST software is suboptimal for very long queries or database sequences. There are also some shortcomings in the user-interface of the current command-line applications. We describe features and improvements of rewritten BLAST software and introduce new command-line applications. Long query sequences are broken into chunks for processing, in some cases leading to dramatically shorter run times. For long database sequences, it is possible to retrieve only the relevant parts of the sequence, reducing CPU time and memory usage for searches of short queries against databases of contigs or chromosomes. The program can now retrieve masking information for database sequences from the BLAST databases. A new modular software library can now access subject sequence data from arbitrary data sources. We introduce several new features, including strategy files that allow a user to save and reuse their favorite set of options. The strategy files can be uploaded to and downloaded from the NCBI BLAST web site. The new BLAST command-line applications, compared to the current BLAST tools, demonstrate substantial speed improvements for long queries as well as chromosome length database sequences. We have also improved the user interface of the command-line applications.

/pdf/blast-architecture-and-applications-2udguz40ns.pdf

BLAST+: architecture and applications.

A 2.91-billion base pair (bp) consensus sequence of the euchromatic portion of the human genome was generated by the whole-genome shotgun sequencing method. The 14.8-billion bp DNA sequence was generated over 9 months from 27,271,853 high-quality sequence reads (5.11-fold coverage of the genome) from both ends of plasmid clones made from the DNA of five individuals. Two assembly strategies-a whole-genome assembly and a regional chromosome assembly-were used, each combining sequence data from Celera and the publicly funded genome effort. The public data were shredded into 550-bp segments to create a 2.9-fold coverage of those genome regions that had been sequenced, without including biases inherent in the cloning and assembly procedure used by the publicly funded group. This brought the effective coverage in the assemblies to eightfold, reducing the number and size of gaps in the final assembly over what would be obtained with 5.11-fold coverage. The two assembly strategies yielded very similar results that largely agree with independent mapping data. The assemblies effectively cover the euchromatic regions of the human chromosomes. More than 90% of the genome is in scaffold assemblies of 100,000 bp or more, and 25% of the genome is in scaffolds of 10 million bp or larger. Analysis of the genome sequence revealed 26,588 protein-encoding transcripts for which there was strong corroborating evidence and an additional approximately 12,000 computationally derived genes with mouse matches or other weak supporting evidence. Although gene-dense clusters are obvious, almost half the genes are dispersed in low G+C sequence separated by large tracts of apparently noncoding sequence. Only 1.1% of the genome is spanned by exons, whereas 24% is in introns, with 75% of the genome being intergenic DNA. Duplications of segmental blocks, ranging in size up to chromosomal lengths, are abundant throughout the genome and reveal a complex evolutionary history. Comparative genomic analysis indicates vertebrate expansions of genes associated with neuronal function, with tissue-specific developmental regulation, and with the hemostasis and immune systems. DNA sequence comparisons between the consensus sequence and publicly funded genome data provided locations of 2.1 million single-nucleotide polymorphisms (SNPs). A random pair of human haploid genomes differed at a rate of 1 bp per 1250 on average, but there was marked heterogeneity in the level of polymorphism across the genome. Less than 1% of all SNPs resulted in variation in proteins, but the task of determining which SNPs have functional consequences remains an open challenge.

http://science.sciencemag.org/content/sci/291/5507/1304.full.pdf

The sequence of the human genome.

Cancer therapies targeting metabolism have been limited due to a lack of understanding of the controlling properties of vulnerable pathways. The Na+/K+ ATPase is responsible for a large portion of cellular energy demands but how these demands influence metabolism and create metabolic liabilities are not known. Using metabolomic approaches, we first show that digoxin, a cardiac glycoside widely used in humans, acts through disruption to central carbon metabolism via on target inhibition of the Na+/K+ ATPase that was fully recovered by expression of an allele resistant to digoxin. We further show in vivo that administration of digoxin inhibits glycolysis in both malignant and healthy cells, particularly within clinically relevant cardiac tissue, while exhibiting tumor-specific cytotoxic activity in an allografted soft tissue sarcoma. Single-cell expression analysis of over 31,000 cells within the sarcoma shows that acute Na+/K+ ATPase inhibition shifts the immune composition of the tumor microenvironment, leading to selective transcriptional reprogramming of metabolic processes in specific immune cells thus acting both through tumor cell autonomous and non-autonomous (e.g. macrophage) cells. These results provide evidence that altering energy demands can be used to regulate glycolysis with specific cell-type specific consequences in a multicellular environment.

/pdf/the-na-k-atpase-regulates-glycolysis-and-defines-8rvx2u2hae.pdf

The Na+/K+ ATPase Regulates Glycolysis and Defines Immunometabolism in Tumors

Profiling serum neurofilament light chain and glial fibrillary acidic protein in primary progressive multiple sclerosis

The identification of genomic loci linked to human disease has been greatly facilitated by the evolution of genotyping strategies and techniques. The success of these strategies continues to be based upon clear clinical assessment, accurate sample handling, and careful data management, but also increasingly upon experimental design. Technological advances in the field of genotyping are permitting increasing complex population studies to be performed. An understanding of publicly available genetic variation databases, including an awareness of the limitations of these data, and an appreciation of the strategic approaches that should be used to exploit this information will provide tremendous insight for researchers are aiming to utilize this increasingly accessible technology. As single-nucleotide polymorphisms become the mainstay of genetic analyses, it is important that their source, distribution and de novo identification before understood before they are incorporated into genetic linkage and association analyses.

Strategies for Genotype Generation

Association of autism with induced or augmented childbirth.

65 Background: Given the evolving treatments available in metastatic castration resistant prostate cancer (mCRPC), predictive biomarkers are desirable that maximize benefit and minimize harms and costs.The goal of this study was to determine the feasibility of DNA copy number and whole exome sequencing (WES) analysis of circulating tumor cells (CTCs) from men with mCRPC receiving enzalutamide. Methods: We collected CTCs from men with mCRPC in the context of enzalutamide therapy. CTCs were isolated from EDTA blood through red cell lysis, CD45 depletion, and flow sorting on EpCAM/CD45 expression. Whole genomic amplification and array based comparative genomic hybridization (CGH) was performed using Qiagen Repli-Gene Single Cell kit, multiple displacement amplification, and Agilent microarray analysis. CTC copy number changes were compared with patient leukocyte DNA and reference metastatic PC datasets. CTC AR amplification and PTEN loss was confirmed with FISH. WES on REPLI-g amplified CTC and leukocyte DNA...

Genomic analysis of circulating tumor cells (CTCs) from men with metastatic castration resistant prostate cancer (mCRPC) in the context of enzalutamide therapy.

Simon G. Gregory

Papers

The Na+/K+ ATPase Regulates Glycolysis and Defines Immunometabolism in Tumors

Profiling serum neurofilament light chain and glial fibrillary acidic protein in primary progressive multiple sclerosis

Strategies for Genotype Generation

Association of autism with induced or augmented childbirth.

Genomic analysis of circulating tumor cells (CTCs) from men with metastatic castration resistant prostate cancer (mCRPC) in the context of enzalutamide therapy.