A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing

Multiplexed high-throughput pyrosequencing is currently limited in complexity (number of samples sequenced in parallel), and in capacity (number of sequences obtained per sample). Physical-space segregation of the sequencing platform into a fixed number of channels allows limited multiplexing, but obscures available sequencing space. To overcome these limitations, we have devised a novel barcoding approach to allow for pooling and sequencing of DNA from independent samples, and to facilitate subsequent segregation of sequencing capacity. Forty-eight forward–reverse barcode pairs are described: each forward and each reverse barcode unique with respect to at least 4 nt positions. With improved read lengths of pyrosequencers, combinations of forward and reverse barcodes may be used to sequence from as many as n² independent libraries for each set of ‘n’ forward and ‘n’ reverse barcodes, for each defined set of cloning-linkers. In two pilot series of barcoded sequencing using the GS20 Sequencer (454/Roche), we found that over 99.8% of obtained sequences could be assigned to 25 independent, uniquely barcoded libraries based on the presence of either a perfect forward or a perfect reverse barcode. The false-discovery rate, as measured by the percentage of sequences with unexpected perfect pairings of unmatched forward and reverse barcodes, was estimated to be <0.005%.     

The interaction of high and low-risk human papillomavirus genotypes increases the risk of developing genital warts: A population-based cohort study

Cervical cancer is among the most common type of cancers in women and is associated with human papillomavirus (HPV) infection. Genital warts are also reported to be linked with HPV infection types 11 and 6. In turn, clinical characteristics and morphological features of warts may be useful in the prediction of prognosis and in making treatment decisions. Thus, we have investigated the association of high and low-risk HPVs genotype with genital wart risk, as well as pathological and cytological information in cases recruited from a population-based cohort study of 1380 patients. Patients infected with HPV genotype 6 or 11 had an increased risk of having warts, with OR of 2.34 (95% CI: 0.955-5.737, P = 0.06). Also, this association was enhanced in the presence of high plus low-risk HPV for having genital wart (OR: 2.814; 95%: 1.208-6.55, P = 0.017) and cases having high-risk HPV (OR: 2.329; 95% CI: 1.029-5.269, P = 0.042). Moreover, we observed patients with genital warts having CIN2/3, indicating the importance of informing the physician to the patient to prevent more severe lesions. Our data demonstrated that patients with both low/high-risk HPV types had an increased risk of developing genital warts and persistent infection with HPV was a necessary precursor for the increase in cervical lesions.     

Analysis of the evolutionary forces in an immunodominant CD8 epitope in hepatitis C virus at a population level

Failure of the adaptive immune response to control infection with the hepatitis C virus (HCV) can result from mutational escape in targeted T-cell epitopes. Recent studies suggest that T-cell immune pressure is an important factor in the evolution of the nonstructural proteins in HCV. The aim of this study was to characterize the forces that contribute to viral evolution in an HLA-A*01-restricted epitope in HCV NS3. This epitope represents a potentially attractive target for vaccination strategies since it is conserved across all genotypes. In our cohort of subjects with chronic HCV infection (genotype 1b or 3a), it is a frequently recognized CD8 epitope in HLA-A*01-positive subjects. Viral sequence data reveal that an escape variant is the dominant residue in both genotypes. The predominant Y1444F substitution seemingly impairs binding to the HLA-A*01 molecule, which may have an important impact on the ability to prime a functional CD8 response upon infection. Interestingly, a case of evolution toward the prototype sequence was observed during chronic infection, possibly because the helicase activity of the protein containing the Y1444F substitution is reduced compared to the prototype sequence. Comparison of HCV sequences from Asia and Europe suggests that the frequency of the HLA-A*01 allele in a population may influence the frequency of the escape variant in circulating strains. These data suggest a complex interaction of multiple forces shaping the evolution of HCV in which immune pressure both within the individual and also at the population level in addition to functional constraints are important contributing factors.     

Quinol type compound in cytochrome c preparations leads to non-enzymatic reduction of cytochrome c during the measurement of complex III activity

Measurement of complex III activity is critical to the diagnosis of human mitochondrial disease and the study of mitochondrial pathobiology. Activity is measured as the maximal rate of antimycin A-sensitive reduction of exogenous cytochrome c by detergent-solubilized mitochondria. Complex III activity exhibited an unexpected variation based upon the commercial source of cytochrome c owing to an increase in the antimycin A-insensitive background reduction of cytochrome c and variable increases in total activity. Analysis of cytochrome c (producing a high-background) by fast protein liquid chromatography yielded a contaminant peak containing a lipid extractable component with redox spectra and mass spectroscopy fragmentation suggestive of a quinol. Measurement of inhibitor-sensitive rates are critical for the accurate and reproducible measurement of complex III activity and serve as a key quality control to screen for non-enzymatic reactions that obscure complex III activity.     

High throughput automated allele frequency estimation by pyrosequencing

Pyrosequencing is a DNA sequencing method based on the principle of sequencing-by-synthesis and pyrophosphate detection through a series of enzymatic reactions. This bioluminometric, real-time DNA sequencing technique offers unique applications that are cost-effective and user-friendly. In this study, we have combined a number of methods to develop an accurate, robust and cost efficient method to determine allele frequencies in large populations for association studies. The assay offers the advantage of minimal systemic sampling errors, uses a general biotin amplification approach, and replaces dTTP for dATP-apha-thio to avoid non-uniform higher peaks in order to increase accuracy. We demonstrate that this newly developed assay is a robust, cost-effective, accurate and reproducible approach for large-scale genotyping of DNA pools. We also discuss potential improvements of the software for more accurate allele frequency analysis.     

A Genetic Variant in Proline and Serine Rich Coiled-Coil 1 Gene Is Associated with the Risk of Cardiovascular Disease

Background:Cardiovascular disease is one of the most common causes of morbidity and mortality worldwide. The Proline and Serine Rich Coiled-Coil 1 gene in 1p13.3 locus has been reported to be associated with low density lipoprotein cholesterol (LDL-C) and coronary artery disease (CAD). The objective of this study was to investigate the association between the rs599839 polymorphism of the Proline and Serine Rich Coiled-Coil 1 (PSRC1) gene with CVD outcomes in a population sample recruited as part of the Mashhad-Stroke and Heart-Atherosclerotic-Disorders (MASHAD) cohort.Methods:Five hundred and nine individuals who had an average follow-up period of 10 years were enrolled as part of the MASHAD cohort. DNA was extracted and genotyped using the TaqMan-real-time-PCR based method.Results:The study found individuals with GA/GG genotypes were at a higher risk of CVDs (OR= 4.7; 95% CI, 2.5-8.7; p< 0.001) in comparison to those with AA genotype; however, the result was not significant for GG genotype data.Conclusion:The results suggest that the GA/GG genotypes of the PSRC1gene locus were at increased risk of CVD in a representative population-based cohort, demonstrating further functional analysis to discover the value of emerging marker as a risk stratification biomarker to recognize high risk cases.Key Words: Cardiovascular diseases, Cohort studies, Genetic Polymorphism, PSRC1 Gene