Next generation sequencing (NGS) technology has revolutionized genomic and genetic research. The pace of change in this area is rapid with three major new sequencing platforms having been released in 2011: Ion Torrent’s PGM, Pacific Biosciences’ RS and the Illumina MiSeq. Here we compare the results obtained with those platforms to the performance of the Illumina HiSeq, the current market leader. In order to compare these platforms, and get sufficient coverage depth to allow meaningful analysis, we have sequenced a set of 4 microbial genomes with mean GC content ranging from 19.3 to 67.7%. Together, these represent a comprehensive range of genome content. Here we report our analysis of that sequence data in terms of coverage distribution, bias, GC distribution, variant detection and accuracy. Sequence generated by Ion Torrent, MiSeq and Pacific Biosciences technologies displays near perfect coverage behaviour on GC-rich, neutral and moderately AT-rich genomes, but a profound bias was observed upon sequencing the extremely AT-rich genome of Plasmodium falciparum on the PGM, resulting in no coverage for approximately 30% of the genome. We analysed the ability to call variants from each platform and found that we could call slightly more variants from Ion Torrent data compared to MiSeq data, but at the expense of a higher false positive rate. Variant calling from Pacific Biosciences data was possible but higher coverage depth was required. Context specific errors were observed in both PGM and MiSeq data, but not in that from the Pacific Biosciences platform. All three fast turnaround sequencers evaluated here were able to generate usable sequence. However there are key differences between the quality of that data and the applications it will support.

/pdf/a-tale-of-three-next-generation-sequencing-platforms-kwlq74rwzb.pdf

A tale of three next generation sequencing platforms: comparison of Ion Torrent, Pacific Biosciences and Illumina MiSeq sequencers.

Genotype imputation is a statistical technique that is often used to increase the power and resolution of genetic association studies. Imputation methods work by using haplotype patterns in a reference panel to predict unobserved genotypes in a study dataset, and a number of approaches have been proposed for choosing subsets of reference haplotypes that will maximize accuracy in a given study population. These panel selection strategies become harder to apply and interpret as sequencing efforts like the 1000 Genomes Project produce larger and more diverse reference sets, which led us to develop an alternative framework. Our approach is built around a new approximation that uses local sequence similarity to choose a custom reference panel for each study haplotype in each region of the genome. This approximation makes it computationally efficient to use all available reference haplotypes, which allows us to bypass the panel selection step and to improve accuracy at low-frequency variants by capturing unexpected allele sharing among populations. Using data from HapMap 3, we show that our framework produces accurate results in a wide range of human populations. We also use data from the Malaria Genetic Epidemiology Network (MalariaGEN) to provide recommendations for imputation-based studies in Africa. We demonstrate that our approximation improves efficiency in large, sequence-based reference panels, and we discuss general computational strategies for modern reference datasets. Genome-wide association studies will soon be able to harness the power of thousands of reference genomes, and our work provides a practical way for investigators to use this rich information. New methodology from this study is implemented in the IMPUTE2 software package.

/pdf/genotype-imputation-with-thousands-of-genomes-4xttelpu21.pdf

Genotype Imputation with Thousands of Genomes

It has been 100 years since the first report of sickle haemoglobin (HbS). More than 50 years ago, it was suggested that the gene responsible for this disorder could reach high frequencies because of resistance conferred against malaria by the heterozygous carrier state. This traditional example of balancing selection is known as the 'malaria hypothesis'. However, the geographical relationship between the transmission intensity of malaria and associated HbS burden has never been formally investigated on a global scale. Here, we use a comprehensive data assembly of HbS allele frequencies to generate the first evidence-based map of the worldwide distribution of the gene in a Bayesian geostatistical framework. We compare this map with the pre-intervention distribution of malaria endemicity, using a novel geostatistical area-mean comparison. We find geographical support for the malaria hypothesis globally; the relationship is relatively strong in Africa but cannot be resolved in the Americas or in Asia.

/pdf/global-distribution-of-the-sickle-cell-gene-and-geographical-53pvwwy722.pdf

Global distribution of the sickle cell gene and geographical confirmation of the malaria hypothesis

Cross-contamination between cell lines is a longstanding and frequent cause of scientific misrepresentation. Estimates from national testing services indicate that up to 36% of cell lines are of a different origin or species to that claimed. To test a standard method of cell line authentication, 253 human cell lines from banks and research institutes worldwide were analyzed by short tandem repeat profiling. The short tandem repeat profile is a simple numerical code that is reproducible between laboratories, is inexpensive, and can provide an international reference standard for every cell line. If DNA profiling of cell lines is accepted and demanded internationally, scientific misrepresentation because of cross-contamination can be largely eliminated.

/pdf/short-tandem-repeat-profiling-provides-an-international-1hqoe0likm.pdf

Short tandem repeat profiling provides an international reference standard for human cell lines

Recent genome-wide studies have reported novel associations between common polymorphisms and susceptibility to many major infectious diseases in humans In parallel, an increasing number of rare mutations underlying susceptibility to specific phenotypes of infectious disease have been described Together, these developments have highlighted a key role for host genetic variation in determining the susceptibility to infectious disease They have also provided insights into the genetic architecture of infectious disease susceptibility and identified immune molecules and pathways that are directly relevant to the human host defence

Human genetic susceptibility to infectious disease

The increasing diversity of new cell cultures is seriously stretching the capabilities of traditional methods of identification. DNA fingerprinting is set to play an important role in increasing confidence in the authenticity of cultures in research and industry.

DNA fingerprinting transforms the art of cell authentication.

The technique of multilocus DNA fingerprinting has great potential for the authentication of animal cell cultures and in identification of cross-contamination. The Alec Jeffreys probes 33.6 and 33.15 were used as multilocus probes to demonstrate the consistent DNA fingerprint profiles in human peripheral blood and its derivative Epstein-Barr virus (EBV) transformed B-lymphoblastoid cultures maintained by repeated subculture for six months. However, fingerprint analysis of EBV transformed cultures generated from small numbers of cells showed that the majority (seven of eight cultures) had anomalous profiles. Some of these altered profiles shared common features not seen in the peripheral blood pattern. Analysis of seven murine hybridoma clones from a single fusion experiment revealed only two clones which could not be distinguished using probe 33.15. Further studies of master and distribution cell banks for eleven cell lines demonstrated consistent fingerprint profiles in all cases except one (U937). However, this cell line showed only minor differences in the master and distribution bank profiles. These data indicate that, while changes in fingerprint profile may be identified in exceptional instances, the multilocus fingerprinting method using probes 33.6 and 33.15 is a powerful and reliable tool in the quality control of animal cell cultures.

Multilocus DNA fingerprint analysis of cellbanks: Stability studies and culture identification in human B-lymphoblastoid and mammalian cell lines

Authentication of Animal Cell Cultures by Direct Visualization of Repetitive DNA, Aldolase Gene PCR and Isoenzyme Analysis

DNA fingerprinting is an important new development for the authentication of cell lines. Multilocus methods such as those developed by Alec Jeffreys provide information on a wide range of genetic loci throughout the human genome and thus give a useful genetic "snap-shot" of a cell culture. Our work has shown that Jeffreys multilocus fingerprinting method can be applied to cell lines from a wide range of animals including reptiles, birds, fish and diverse mammals. It can also differentiate very closely related cell lines including those from the same mouse strain. Routine fingerprint analysis has enabled an unprecedented level of confidence in the consistency of cell stocks. Our results demonstrate that this straightforward method represents a powerful and readily interpreted system for cell authentication and exclusion of cross-contamination.

DNA fingerprinting--a valuable new technique for the characterisation of cell lines.

List of Contributors. Preface by the Editor. 1. Standardisation for in vitro Toxicity Test M. Ferro, A. Doyle. 2. Application of Cell Cultures to Toxicology I. Freshney. 3. Immortalisation of Primary Cells G. Stacey, C. MacDonald. 4. Safety Considerations for in vitro Toxicology Testing B. Jones, G. Stacey. 5. The Drug Metabolizing Enzymatic System and the Experimental Tools Used for in vitro Toxicology for Metabolic Studies E. Testai. 6. Cell Culture Models for Hepatotoxicology T. Battle, G. Stacey. 7. Intestinal Cell Culture Models: Applications of Toxicology and Pharmacology Y. Sambruy, S. Ferruzza, G. Ranaldi, I. de Angelis. 8. Cell Culture Models for Neurotoxicology G. Stacey, B. Viviani. 9. Recombinant Cell Lines for Stress Reporter Assays M. Fischbach, P. Bromley. Index.

Cell culture methods for in vitro toxicology

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