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The dynamics of emerging nucleoside and nucleotide reverse-transcriptase inhibitor (NRTI) resistance in hepatitis B virus (HBV) are not well understood because standard dideoxynucleotide direct polymerase chain reaction (PCR) sequencing assays detect drug-resistance mutations only after they have become dominant. To obtain insight into NRTI resistance, we used a new sequencing technology to characterize the spectrum of low-prevalence NRTI-resistance mutations in HBV obtained from 20 plasma samples from 11 NRTI-treated patients and 17 plasma samples from 17 NRTI-naive patients, by using standard direct PCR sequencing and ultra-deep pyrosequencing (UDPS). UDPS detected drug-resistance mutations that were not detected by PCR in 10 samples from 5 NRTI-treated patients, including the lamivudine-resistance mutation V173L (in 5 samples), the entecavir-resistance mutations T184S (in 2 samples) and S202G (in 1 sample), the adefovir-resistance mutation N236T (in 1 sample), and the lamivudine and adefovir-resistance mutations V173L, L180M, A181T, and M204V (in 1 sample). G-to-A hypermutation mediated by the apolipoprotein B mRNA editing enzyme, catalytic polypeptide-like family of cytidine deaminases was estimated to be present in 0.6% of reverse-transcriptase genes. Genotype A coinfection was detected by UDPS in each of 3 patients in whom genotype G virus was detected by direct PCR sequencing. UDPS detected low-prevalence HBV variants with NRTI-resistance mutations, G-to-A hypermutation, and low-level dual genotype infection with a sensitivity not previously possible.

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 n(2) 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%.

Context: The polymorphism of class II HLA genes modulates the genetic risk for several endocrine autoimmune diseases. The constitutive class II expression on antigen-presenting cells is under the control of the MHC class II transactivator, encoded by the MHC2TA gene, which is mapped to chromosome 16p13. The MHC2TA -168 A-->G single nucleotide polymorphism (rs3087456) has been suggested to confer susceptibility to some autoimmune diseases., Design: With the aim of testing whether this MHC2TA single nucleotide polymorphism is independently associated with autoimmune Addison's disease (AAD) and/or modulates the genetic risk conferred by DRB1-DQA1-DQB1 haplotypes, we analyzed DNA samples from 128 AAD patients and 406 healthy control subjects from continental Italy., Results: Frequency of allele G of MHC2TA was significantly increased among AAD patients (39% alleles), compared with 29% in healthy controls (P = 0.003). Similarly, the frequency of AG+GG genotypes was significantly higher among AAD patients than among healthy control subjects, in both a codominant (P = 0.012) and a G-dominant model (P = 0.018). Multivariate logistic regression analysis showed that MHC2TA AG+GG continued to be positively associated with genetic risk for AAD (P = 0.028, odds ratio = 1.72, 95% confidence interval = 1.06-2.78), after correction for DRB1*03-DQA1*0501-DQB1*0201, DRB1*04 (not 0403)-DQA1*0301-DQB1*0302 and DRB1*0403. Similar results were obtained when the number of G alleles was included in the model (P = 0.004; odds ratio = 1.65, 95% confidence interval = 1.17-2.32)., Conclusions: Our study provides the first demonstration of the association of the polymorphism of the MHC2TA gene with genetic risk for AAD that appears to be independent from the well-known association with the polymorphism of HLA class II genes.

Detection and identification of microbial pathogens are important for disease diagnosis, treatment and prophylaxis measurements. By introducing an innovative technique, we show a robust, reliable and accurate microarray-based method for identification of microbial pathogens. The technique utilizes a unique combination of multiplex competitive hybridization, which enhances hybridization accuracy of oligonucleotides to the specific target, and apyrase-mediated allele-specific extension, which improves specific extension. As a model system, different clinically relevant human papillomaviruses were selected for this study. The method generated accurate results and proves to be promising for specific and correct microbial and viral typing.

This report describes a single-step extension approach suitable for high-throughput single-nucleotide polymorphism typing applications. The method relies on extension of paired allele-specific primers and we demonstrate that the reaction kinetics were slower for mismatched configurations compared with matched configurations. In our approach we employ apyrase, a nucleotide degrading enzyme, to allow accurate discrimination between matched and mismatched primer-template configurations. This apyrase-mediated allele-specific extension (AMASE) protocol allows incorporation of nucleotides when the reaction kinetics are fast (matched 3'-end primer) but degrades the nucleotides before extension when the reaction kinetics are slow (mismatched 3'-end primer). Thus, AMASE circumvents the major limitation of previous allele-specific extension assays in which slow reaction kinetics will still give rise to extension products from mismatched 3'-end primers, hindering proper discrimination. It thus represents a significant improvement of the allele-extension method. AMASE was evaluated by a bioluminometric assay in which successful incorporation of unmodified nucleotides is monitored in real-time using an enzymatic cascade.