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

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.In recent years, there has been a rapid expansion in the number of next-generation sequencing platforms, including Illumina (Bentley et al. 2008), the Applied Biosystems SOLiD System (McKernan et al. 2009), 454 Life Sciences (Roche) (Margulies et al. 2005), Helicos HeliScope (Shendure and Ji 2008), and most recently Complete Genomics (Drmanac et al. 2010). Many tools have been created to work with next-generation sequencer data, from read based aligners like MAQ (Li et al. 2008a), BWA (Li and Durbin 2009), and SOAP (Li et al. 2008b), to single nucleotide polymorphism and structural variation detection tools like BreakDancer (Chen et al. 2009), VarScan (Koboldt et al. 2009), and MAQ. Although these tools are highly effective in their problem domains, there still exists a large development gap between sequencing output and analysis results, in part because tailoring these analysis tools to answer specific scientific questions can be laborious and difficult. General frameworks are available for processing next-generation sequencing data but tend to focus on specific classes of analysis problems—like quality assessment of sequencing data, as in PIQA (Martinez-Alcantara et al. 2009)—or require specialized knowledge of an existing framework, as in BioConductor in the ShortRead toolset (Morgan et al. 2009). The lack of sophisticated and flexible programming frameworks that enable downstream analysts to access and manipulate the massive sequencing data sets in a programmatic way has been a hindrance to the rapid development of new tools and methods.With the emergence of the SAM file specification (Li et al. 2009) as the standard format for storage of platform-independent next-generation sequencing data, we saw the opportunity to implement an analysis programming framework which takes advantage of this common input format to simplify the up-front coding costs for end users. Here, we present the 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 (Dean and Ghemawat 2008). By separating specific analysis calculations from common data management infrastructure, tools are easy to write while benefiting from ongoing improvements to the core GATK. The GATK engine is constantly being refined and optimized for correctness, stability, and CPU and memory efficiency; this well-structured software core allows the GATK to support advanced features such as distributed and automatic shared-memory parallelization. Here, we highlight the capabilities of the GATK, which has been used to implement a range of analysis methods for projects like The Cancer Genome Atlas (http://cancergenome.nih.gov) and the 1000 Genomes Project (http://www.1000genomes.org), by describing the implementation of depth of coverage analysis tools and a Bayesian single nucleotide polymorphism (SNP) genotyper, and show the application of these tools to the 1000 Genomes Project pilot data.     

Relapsing eosinophilic perimyositis

Relapsing eosinophilic perimyositis is a rare entity. Only 11 cases have been reported in the literature. We describe a patient with relapsing myopathy, peripheral blood eosinophilia and cutaneous manifestations, who had histopathological features of eosinophilic perimysial inflammation. His disease responded to moderate doses of glucocorticosteroids, and pursued an apparently benign course. Serum levels of eosinophilic cationic protein correlate with disease activity, and may be helpful in monitoring treatment. The features of this case are compared with those of other hypereosinophilic syndromes. It appears to be part of the spectrum of eosinophilic myositis.     

Gianotti-Crosti syndrome: a study of 26 cases

We have studied 26 patients presenting with a symmetrical papular or papulovesicular acrolocated eruption of more than 10 days duration. Mean age at onset was 2 years (range 10 months to 5.75 years). Lymphadenopathy was noted in eight cases, and hepatomegaly in one case. In 12 cases, histopathology and direct immunofluorescence were non-contributory. Cytolytic hepatitis occurred in one case and was associated with HBs antigenemia. A history of recent immunization was given in two cases. There was serological evidence of recent Epstein-Barr virus infection in seven out of 13 cases tested. Coxsackie B viruses were isolated from three patients, and cytomegalovirus was probably associated with the syndrome in one case. We conclude that the Gianotti-Crosti syndrome is not rare in France, and that non-hepatitis B virus (HBV)-associated cases are more frequent than the classical HBV-associated papular acrodermatitis of childhood.     

Platelet membrane microparticles in blood bank fresh frozen plasma and cryoprecipitate

Cryoprecipitate has been demonstrated to correct the bleeding abnormality of patients with some congenital (storage pool disease) and acquired (uremia) platelet abnormalities, but the reason for this effect is unknown. We found significant platelet contamination in plasma harvested to prepare fresh frozen plasma and cryoprecipitate. The platelet membrane microparticles produced by freezing and thawing of the plasma were highly concentrated in cryoprecipitate and may contribute to its therapeutic effect.     

Hepatobiliary scintigraphy in the diagnosis of gallstones in young people

The article describes results of using dynamic hepatobiliary scintigraphy in 43 young patients with chronic calculous cholecystitis. High informative value of the method for diagnosis of morpho-functional changes in the organs of the hepatobiliary system is shown, hyperbilirubinemia included. More than in half of the patients alterations of the absorptive-excretory functions of the liver and motor function of the gallbladder were revealed. Their direct relationship with the duration of the disease and degree of the inflammatory process in the biliary tract is established.     

Risk factors for early termination of breast feeding in Brazil

A prospective study was undertaken to identify possible factors related to the duration of breast feeding. Two hundred and thirty-eight mothers who had delivered normal single babies with birth weights greater than 2.5 kg and had initiated breast feeding were randomly selected at the maternity hospital, Hospital de Clinicas de Porto Alegre, Brazil, and followed by mail questionnaires until termination of breast feeding, or until the end of the first year. If no reply was received, telephone contact or home visits were made. The group of mothers who stopped breast feeding prior to the end of the third month was compared with those who extended breast feeding beyond three months with respect to socioeconomic, biological, environmental, medical and psychological factors. The variables with a significant coefficient of association with early termination of breast feeding were maternal education, past experience with breast feeding, help of a maid, help with housework provided by a relative, breast feeding orientation during prenatal care and encouragement from the husband. These factors act simultaneously, with interactions among them.     

Bone marrow immunophenotyping for the diagnosis of multiple myeloma: practical aspects

Multiple myeloma is a malignant proliferative disease of plasma cells. Flow cytometric immunophenotyping makes it possible to identify a malignant clone of myeloma cells in the shortest possible time, to determine its phenotype, and differentiate transformed and preserved plasma cells. The article presents an immunophenotyping strategy using three-color monoclonal antibodies (CD35, CD14, CD38, CD138, and CD19) and an algorithm of verification of transformed plasma cells. Particular emphasis is placed on both the practical aspects of performing this assay and on the clinical application of the obtained results.