Next-generation sequencing technologies and applications for human genetic history and forensics

Rapid advances in the development of sequencing technologies in recent years have enabled an increasing number of applications in biology and medicine. Here, we review key technical aspects of the preparation of DNA templates for sequencing, the biochemical reaction principles and assay formats underlying next-generation sequencing systems, methods for imaging and base calling, quality control, and bioinformatic approaches for sequence alignment, variant calling and assembly. We also discuss some of the most important advances that the new sequencing technologies have brought to the fields of human population genetics, human genetic history and forensic genetics

Silhouette scores for assessment of SNP genotype clusters

BACKGROUND: High-throughput genotyping of single nucleotide polymorphisms (SNPs) generates large amounts of data. In many SNP genotyping assays, the genotype assignment is based on scatter plots of signals corresponding to the two SNP alleles. In a robust assay the three clusters that define the genotypes are well separated and the distances between the data points within a cluster are short. "Silhouettes" is a graphical aid for interpretation and validation of data clusters that provides a measure of how well a data point was classified when it was assigned to a cluster. Thus "Silhouettes" can potentially be used as a quality measure for SNP genotyping results and for objective comparison of the performance of SNP assays at different circumstances. RESULTS: We created a program (ClusterA) for calculating "Silhouette scores", and applied it to assess the quality of SNP genotype clusters obtained by single nucleotide primer extension ("minisequencing") in the Tag-microarray format. A Silhouette score condenses the quality of the genotype assignment for each SNP assay into a single numeric value, which ranges from 1.0, when the genotype assignment is unequivocal, down to -1.0, when the genotype assignment has been arbitrary. In the present study we applied Silhouette scores to compare the performance of four DNA polymerases in our minisequencing system by analyzing 26 SNPs in both DNA polarities in 16 DNA samples. We found Silhouettes to provide a relevant measure for the quality of SNP assays at different reaction conditions, illustrated by the four DNA polymerases here. According to our result, the genotypes can be unequivocally assigned without manual inspection when the Silhouette score for a SNP assay is > 0.65. All four DNA polymerases performed satisfactorily in our Tag-array minisequencing system. CONCLUSION: "Silhouette scores" for assessing the quality of SNP genotyping clusters is convenient for evaluating the quality of SNP genotype assignment, and provides an objective, numeric measure for comparing the performance of SNP assays. The program we created for calculating Silhouette scores is freely available, and can be used for quality assessment of the results from all genotyping systems, where the genotypes are assigned by cluster analysis using scatter plots

Detecting imbalanced expression of SNP alleles by minisequencing on microarrays

BACKGROUND: Each of the human genes or transcriptional units is likely to contain single nucleotide polymorphisms that may give rise to sequence variation between individuals and tissues on the level of RNA. Based on recent studies, differential expression of the two alleles of heterozygous coding single nucleotide polymorphisms (SNPs) may be frequent for human genes. Methods with high accuracy to be used in a high throughput setting are needed for systematic surveys of expressed sequence variation. In this study we evaluated two formats of multiplexed, microarray based minisequencing for quantitative detection of imbalanced expression of SNP alleles. We used a panel of ten SNPs located in five genes known to be expressed in two endothelial cell lines as our model system. RESULTS: The accuracy and sensitivity of quantitative detection of allelic imbalance was assessed for each SNP by constructing regression lines using a dilution series of mixed samples from individuals of different genotype. Accurate quantification of SNP alleles by both assay formats was evidenced for by R(2 )values > 0.95 for the majority of the regression lines. According to a two sample t-test, we were able to distinguish 1–9% of a minority SNP allele from a homozygous genotype, with larger variation between SNPs than between assay formats. Six of the SNPs, heterozygous in either of the two cell lines, were genotyped in RNA extracted from the endothelial cells. The coefficient of variation between the fluorescent signals from five parallel reactions was similar for cDNA and genomic DNA. The fluorescence signal intensity ratios measured in the cDNA samples were compared to those in genomic DNA to determine the relative expression levels of the two alleles of each SNP. Four of the six SNPs tested displayed a higher than 1.4-fold difference in allelic ratios between cDNA and genomic DNA. The results were verified by allele-specific oligonucleotide hybridisation and minisequencing in a microtiter plate format. CONCLUSIONS: We conclude that microarray based minisequencing is an accurate and accessible tool for multiplexed screening for imbalanced allelic expression in multiple samples and tissues in parallel

Single nucleotide polymorphisms in the apolipoprotein B and low density lipoprotein receptor genes affect response to antihypertensive treatment

BACKGROUND: Dyslipidemia has been associated with hypertension. The present study explored if polymorphisms in genes encoding proteins in lipid metabolism could be used as predictors for the individual response to antihypertensive treatment. METHODS: Ten single nucleotide polymorphisms (SNP) in genes related to lipid metabolism were analysed by a microarray based minisequencing system in DNA samples from ninety-seven hypertensive subjects randomised to treatment with either 150 mg of the angiotensin II type 1 receptor blocker irbesartan or 50 mg of the β(1)-adrenergic receptor blocker atenolol for twelve weeks. RESULTS: The reduction in blood pressure was similar in both treatment groups. The SNP C711T in the apolipoprotein B gene was associated with the blood pressure response to irbesartan with an average reduction of 19 mmHg in the individuals carrying the C-allele, but not to atenolol. The C16730T polymorphism in the low density lipoprotein receptor gene predicted the change in systolic blood pressure in the atenolol group with an average reduction of 14 mmHg in the individuals carrying the C-allele. CONCLUSIONS: Polymorphisms in genes encoding proteins in the lipid metabolism are associated with the response to antihypertensive treatment in a drug specific pattern. These results highlight the potential use of pharmacogenetics as a guide for individualised antihypertensive treatment, and also the role of lipids in blood pressure control

Coffee consumption and CYP1A2 genotype in relation to bone mineral density of the proximal femur in elderly men and women: a cohort study

ABSTRACT: BACKGROUND: Drinking coffee has been linked to reduced calcium conservation, but it is less clear whether it leads to sustained bone mineral loss and if individual predisposition for caffeine metabolism might be important in this context. Therefore, the relation between consumption of coffee and bone mineral density (BMD) at the proximal femur in men and women was studied, taking into account, for the first time, genotypes for cytochrome P450 1A2 (CYP1A2) associated with metabolism of caffeine. METHODS: Dietary intakes of 359 men and 358 women (aged 72 years), participants of the Prospective Investigation of the Vasculature in Uppsala Seniors (PIVUS), were assessed by a 7-day food diary. Two years later, BMD for total proximal femur, femoral neck and trochanteric regions of the proximal femur were measured by Dual-energy X-ray absorptiometry (DXA). Genotypes of CYP1A2 were determined. Adjusted means of BMD for each category of coffee consumption were calculated. RESULTS: Men consuming 4 cups of coffee or more per day had 4% lower BMD at the proximal femur (p = 0.04) compared with low or non-consumers of coffee. This difference was not observed in women. In high consumers of coffee, those with rapid metabolism of caffeine (C/C genotype) had lower BMD at the femoral neck (p = 0.01) and at the trochanter (p = 0.03) than slow metabolizers (T/T and C/T genotypes). Calcium intake did not modify the relation between coffee and BMD. CONCLUSION: High consumption of coffee seems to contribute to a reduction in BMD of the proximal femur in elderly men, but not in women. BMD was lower in high consumers of coffee with rapid metabolism of caffeine, suggesting that rapid metabolizers of caffeine may constitute a risk group for bone loss induced by coffee

Performance of Microarray and Liquid Based Capture Methods for Target Enrichment for Massively Parallel Sequencing and SNP Discovery

Targeted sequencing is a cost-efficient way to obtain answers to biological questions in many projects, but the choice of the enrichment method to use can be difficult. In this study we compared two hybridization methods for target enrichment for massively parallel sequencing and single nucleotide polymorphism (SNP) discovery, namely Nimblegen sequence capture arrays and the SureSelect liquid-based hybrid capture system. We prepared sequencing libraries from three HapMap samples using both methods, sequenced the libraries on the Illumina Genome Analyzer, mapped the sequencing reads back to the genome, and called variants in the sequences. 74–75% of the sequence reads originated from the targeted region in the SureSelect libraries and 41–67% in the Nimblegen libraries. We could sequence up to 99.9% and 99.5% of the regions targeted by capture probes from the SureSelect libraries and from the Nimblegen libraries, respectively. The Nimblegen probes covered 0.6 Mb more of the original 3.1 Mb target region than the SureSelect probes. In each sample, we called more SNPs and detected more novel SNPs from the libraries that were prepared using the Nimblegen method. Thus the Nimblegen method gave better results when judged by the number of SNPs called, but this came at the cost of more over-sampling

Genetic analysis of an F2 intercross between two chicken lines divergently selected for body-weight

<p>Abstract</p> <p>Background</p> <p>We have performed Quantitative Trait Loci (QTL) analysis of an F₂intercross between two chicken lines divergently selected for juvenile body-weight. In a previous study 13 identified loci with effects on body-weight, only explained a small proportion of the large variation in the F₂population. Epistatic interaction analysis however, indicated that a network of interacting loci with large effect contributed to the difference in body-weight of the parental lines. This previous analysis was, however, based on a sparse microsatellite linkage map and the limited coverage could have affected the main conclusions. Here we present a revised QTL analysis based on a high-density linkage map that provided a more complete coverage of the chicken genome. Furthermore, we utilized genotype data from ~13,000 SNPs to search the genome for potential selective sweeps that have occurred in the selected lines.</p> <p>Results</p> <p>We constructed a linkage map comprising 434 genetic markers, covering 31 chromosomes but leaving seven microchromosomes uncovered. The analysis showed that seven regions harbor QTL that influence growth. The pair-wise interaction analysis identified 15 unique QTL pairs and notable is that nine of those involved interactions with a locus on chromosome 7, forming a network of interacting loci. The analysis of ~13,000 SNPs showed that a substantial proportion of the genetic variation present in the founder population has been lost in either of the two selected lines since ~60% of the SNPs polymorphic among lines showed fixation in one of the lines. With the current marker coverage and QTL map resolution we did not observe clear signs of selective sweeps within QTL intervals.</p> <p>Conclusion</p> <p>The results from the QTL analysis using the new improved linkage map are to a large extent in concordance with our previous analysis of this pedigree. The difference in body-weight between the parental chicken lines is caused by many QTL each with a small individual effect. Although the increased chromosomal marker coverage did not lead to the identification of additional QTL, we were able to refine the localization of QTL. The importance of epistatic interaction as a mechanism contributing significantly to the remarkable selection response was further strengthened because additional pairs of interacting loci were detected with the improved map.</p

Allelic expression mapping across cellular lineages to establish impact of non-coding SNPs

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Association of NCF2, IKZF1, IRF8, IFIH1, and TYK2 with Systemic Lupus Erythematosus

Systemic lupus erythematosus (SLE) is a complex trait characterised by the production of a range of auto-antibodies and a diverse set of clinical phenotypes. Currently, ∼8% of the genetic contribution to SLE in Europeans is known, following publication of several moderate-sized genome-wide (GW) association studies, which identified loci with a strong effect (OR>1.3). In order to identify additional genes contributing to SLE susceptibility, we conducted a replication study in a UK dataset (870 cases, 5,551 controls) of 23 variants that showed moderate-risk for lupus in previous studies. Association analysis in the UK dataset and subsequent meta-analysis with the published data identified five SLE susceptibility genes reaching genome-wide levels of significance (Pcomb<5×10−8): NCF2 (Pcomb = 2.87×10−11), IKZF1 (Pcomb = 2.33×10−9), IRF8 (Pcomb = 1.24×10−8), IFIH1 (Pcomb = 1.63×10−8), and TYK2 (Pcomb = 3.88×10−8). Each of the five new loci identified here can be mapped into interferon signalling pathways, which are known to play a key role in the pathogenesis of SLE. These results increase the number of established susceptibility genes for lupus to ∼30 and validate the importance of using large datasets to confirm associations of loci which moderately increase the risk for disease

L’utilisation des réseaux sociaux (Snapchat, WhatsApp et Instagram) et le cyberbullying

100% des jeunes possèdent un téléphone portable, 99% ont un ordinateur et 97% ont accès à Internet (Waller et al., 2016). Ces nouveaux moyens technologiques font partie de notre quotidien. Depuis l’apparition de ces réseaux, un nouveau mouvement est né : le cyberbullying. Ce harcèlement par Internet consiste à l’utilisation de technologies modernes de communication afin de nuire aux autres de manière délibérée et agressive. Quand les jeunes arrivent en classe, ils apportent avec eux l’entier de leur vécu quotidien, familial ou encore émotionnel. Les problèmes liés à l’utilisation massive de ces réseaux font partie de notre quotidien d’enseignant. Malheureusement, les études faites jusqu’au jour d’aujourd’hui portent en majeure partie sur les élèves entre 13 ans et plus. Mais qu’en est-il des jeunes âgés entre 9 et 12 ans ? Notre travail de recherche porte donc sur l’utilisation des réseaux sociaux (Snapchat, Instagram et WhatsApp) et le cyberbullying. Deux outils différents ont été utilisés lors de cette recherche : des questionnaires afin d’avoir des résultats quantitatifs et deux entretiens afin d’avoir un point de vue qualitatif. Nos résultats montrent que WhatsApp est le réseau social le plus utilisé, suivi d’Instagram en deuxième position et finalement de Snapchat. Les élèves considèrent le nombre de dangers et de conflits sur les réseaux comme très faibles. Ils avouent tout de même donner plus d’informations personnelles sur WhatsApp que sur les autres réseaux choisis dans l’étude. Concernant leur vision du contrôle des parents, ils l’estiment très faible. Cependant, il s’agit uniquement de leur avis, il serait intéressant de savoir la réalité des faits en interrogeant les parents. Les deux sujets interrogés savent définir le cyberbullying et connaissent les différents acteurs agissant au sein de cette forme de harcèlement. Ils sont également conscients des différents risques, conséquences ou sentiments que peut ressentir une cyber-victime mais n’abordent pas du tout ceux concernant le témoin ou le cyber-harceleur. En conclusion, notre recherche montre que les réseaux sociaux font partie intégrante du quotidien d’un grand nombre d’élèves. Il est donc essentiel que les enseignants s’interrogent sur les moyens de gérer les problèmes que ceux-ci peuvent amener en classe mais également les moyens de les éviter