A framework for variation discovery and genotyping using next-generation DNA sequencing data

Recent advances in sequencing technology make it possible to comprehensively catalog genetic variation in population samples, creating a foundation for understanding human disease, ancestry and evolution. The amounts of raw data produced are prodigious, and many computational steps are required to translate this output into high-quality variant calls. We present a unified analytic framework to discover and genotype variation among multiple samples simultaneously that achieves sensitive and specific results across five sequencing technologies and three distinct, canonical experimental designs. Our process includes (i) initial read mapping; (ii) local realignment around indels; (iii) base quality score recalibration; (iv) SNP discovery and genotyping to find all potential variants; and (v) machine learning to separate true segregating variation from machine artifacts common to next-generation sequencing technologies. We here discuss the application of these tools, instantiated in the Genome Analysis Toolkit, to deep whole-genome, whole-exome capture and multi-sample low-pass (～4×) 1000 Genomes Project datasets.     

Exome sequencing identifies GRIN2A as frequently mutated in melanoma

The incidence of melanoma is increasing more than any other cancer, and knowledge of its genetic alterations is limited. To systematically analyze such alterations, we performed whole-exome sequencing of 14 matched normal and metastatic tumor DNAs. Using stringent criteria, we identified 68 genes that appeared to be somatically mutated at elevated frequency, many of which are not known to be genetically altered in tumors. Most importantly, we discovered that TRRAP harbored a recurrent mutation that clustered in one position (p. Ser722Phe) in 6 out of 167 affected individuals (～4%), as well as a previously unidentified gene, GRIN2A, which was mutated in 33% of melanoma samples. The nature, pattern and functional evaluation of the TRRAP recurrent mutation suggest that TRRAP functions as an oncogene. Our study provides, to our knowledge, the most comprehensive map of genetic alterations in melanoma to date and suggests that the glutamate signaling pathway is involved in this disease.     

Conserved noncoding sequences are selectively constrained and not mutation cold spots

Noncoding genetic variants are likely to influence human biology and disease, but recognizing functional noncoding variants is difficult. Approximately 3% of noncoding sequence is conserved among distantly related mammals, suggesting that these evolutionarily conserved noncoding regions (CNCs) are selectively constrained and contain functional variation. However, CNCs could also merely represent regions with lower local mutation rates. Here we address this issue and show that CNCs are selectively constrained in humans by analyzing HapMap genotype data. Specifically, new (derived) alleles of SNPs within CNCs are rarer than new alleles in nonconserved regions (P = 3 x 10(-18)), indicating that evolutionary pressure has suppressed CNC-derived allele frequencies. Intronic CNCs and CNCs near genes show greater allele frequency shifts, with magnitudes comparable to those for missense variants. Thus, conserved noncoding variants are more likely to be functional. Allele frequency distributions highlight selectively constrained genomic regions that should be intensively surveyed for functionally important variation.     

Association of a functional variant downstream of TNFAIP3 with systemic lupus erythematosus

Systemic lupus erythematosus (SLE, MIM152700) is an autoimmune disease characterized by self-reactive antibodies resulting in systemic inflammation and organ failure. TNFAIP3, encoding the ubiquitin-modifying enzyme A20, is an established susceptibility locus for SLE. By fine mapping and genomic re-sequencing in ethnically diverse populations, we fully characterized the TNFAIP3 risk haplotype and identified a TT>A polymorphic dinucleotide (deletion T followed by a T to A transversion) associated with SLE in subjects of European (P = 1.58 × 10(-8), odds ratio = 1.70) and Korean (P = 8.33 × 10(-10), odds ratio = 2.54) ancestry. This variant, located in a region of high conservation and regulatory potential, bound a nuclear protein complex composed of NF-κB subunits with reduced avidity. Further, compared with the non-risk haplotype, the haplotype carrying this variant resulted in reduced TNFAIP3 mRNA and A20 protein expression. These results establish this TT>A variant as the most likely functional polymorphism responsible for the association between TNFAIP3 and SLE.     

Digestive physiology is a determinant of foraging bout frequency in hummingbirds

Hummingbirds are among the smallest endothermic vertebrates. Because they forage by energetically costly hovering, and because weight-specific basal metabolic rates increase with decreasing body size, their basal and active metabolic rates are among the highest recorded. Hummingbirds fuel these metabolic requirements mainly with highly concentrated sugar in nectar, which they extract rapidly and efficiently by an unknown mechanism. It is especially puzzling that, despite their high energy requirements, hummingbirds spend only approximately 20% of their waking hours feeding, but 75% perched and apparently doing nothing. Here we report the first measurement of nutrient absorption by hummingbird intestine and present a new method for measuring crop-emptying times. We find that hummingbird intestine has the highest active glucose transport rate and lowest passive glucose permeability reported for any vertebrate. Crop-emptying time may limit feeding-bout frequency and could largely account for the time spent perched.     

2'-O methylation of the viral mRNA cap evades host restriction by IFIT family members

Cellular messenger RNA (mRNA) of higher eukaryotes and many viral RNAs are methylated at the N-7 and 2'-O positions of the 5' guanosine cap by specific nuclear and cytoplasmic methyltransferases (MTases), respectively. Whereas N-7 methylation is essential for RNA translation and stability, the function of 2'-O methylation has remained uncertain since its discovery 35 years ago. Here we show that a West Nile virus (WNV) mutant (E218A) that lacks 2'-O MTase activity was attenuated in wild-type primary cells and mice but was pathogenic in the absence of type I interferon (IFN) signalling. 2'-O methylation of viral RNA did not affect IFN induction in WNV-infected fibroblasts but instead modulated the antiviral effects of IFN-induced proteins with tetratricopeptide repeats (IFIT), which are interferon-stimulated genes (ISGs) implicated in regulation of protein translation. Poxvirus and coronavirus mutants that lacked 2'-O MTase activity similarly showed enhanced sensitivity to the antiviral actions of IFN and, specifically, IFIT proteins. Our results demonstrate that the 2'-O methylation of the 5' cap of viral RNA functions to subvert innate host antiviral responses through escape of IFIT-mediated suppression, and suggest an evolutionary explanation for 2'-O methylation of cellular mRNA: to distinguish self from non-self RNA. Differential methylation of cytoplasmic RNA probably serves as an example for pattern recognition and restriction of propagation of foreign viral RNA in host cells.