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1.  The Shaping of Modern Human Immune Systems by Multiregional Admixture with Archaic Humans 
Science (New York, N.Y.)  2011;334(6052):89-94.
Whole genome comparisons identified introgression from archaic to modern humans. Our analysis of highly polymorphic HLA class I, vital immune system components subject to strong balancing selection, shows how modern humans acquired the HLA-B*73 allele in west Asia through admixture with archaic humans called Denisovans, a likely sister group to the Neandertals. Virtual genotyping of Denisovan and Neandertal genomes identified archaic HLA haplotypes carrying functionally distinctive alleles that have introgressed into modern Eurasian and Oceanian populations. These alleles, of which several encode unique or strong ligands for natural killer cell receptors, now represent more than half the HLA alleles of modern Eurasians and also appear to have been later introduced into Africans. Thus, adaptive introgression of archaic alleles has significantly shaped modern human immune systems.
doi:10.1126/science.1209202
PMCID: PMC3677943  PMID: 21868630
2.  Next Generation Gene Synthesis by targeted retrieval of bead-immobilized, sequence verified DNA clones from a high throughput pyrosequencing device 
Nature biotechnology  2010;28(12):1291-1294.
The setup of synthetic biological systems involving millions of bases is still limited by the required high quality of synthetic DNA. Important drivers to further open up the field are the accuracy and scale of chemical DNA synthesis and the downstream processing of longer DNA assembled from short fragments. We developed a new, highly parallel and miniaturized method for the preparation of high quality DNA termed “Megacloning” by using Next Generation Sequencing (NGS) technology in a preparative way. We demonstrate our method by processing both conventional and microarray-derived DNA oligonucleotides in combination with a bead-based high throughput pyrosequencing platform, gaining a 500-fold error reduction for microarray oligonucleotides in a first embodiment. We also show the assembly of synthetic genes as part of the Megacloning process. In principle, up to millions of DNA fragments can be sequenced, characterized and sorted in a single Megacloner run, enabling many new applications.
doi:10.1038/nbt.1710
PMCID: PMC3579223  PMID: 21113166
3.  Ultra-Deep Pyrosequencing of Hepatitis B Virus Quasispecies from Nucleoside and Nucleotide Reverse-Transcriptase Inhibitor (NRTI)–Treated Patients and NRTI-Naive Patients 
The Journal of Infectious Diseases  2009;199(9):1275-1285.
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.
doi:10.1086/597808
PMCID: PMC3353721  PMID: 19301976
4.  Although Divergent in Residues of the Peptide-Binding Site, Conserved Chimpanzee Patr-AL and Polymorphic Human HLA-A*02 have Overlapping Peptide-Binding Repertoires 1 
Patr-AL is an expressed, non-polymorphic MHC class I gene carried by ∼50% of chimpanzee MHC haplotypes. Comparing Patr-AL+ and Patr-AL- haplotypes showed Patr-AL defines a unique 125kb genomic block flanked by blocks containing classical Patr-A and pseudogene Patr-H. Orthologous to Patr-AL are polymorphic orangutan Popy-A and the 5′ part of human pseudogene HLA-Y, carried by ∼10% of HLA haplotypes. Thus the AL gene alternatively evolved in these closely related species to become classical, non-classical and non-functional. Although differing by 30 amino acid substitutions in the peptide-binding α1 and α2 domains, Patr-AL and HLA-A*0201 bind overlapping repertoires of peptides; the overlap being comparable to that between the A*0201 and A*0207 subtypes differing by one substitution. Patr-AL thus has the A02 supertypic peptide-binding specificity. Patr-AL and HLA-A*0201 have similar three-dimensional structures, binding peptides in similar conformation. Although comparable in size and shape, the B and F specificity pockets of Patr-AL and HLA-A*0201 differ in both their constituent residues and contacts with peptide anchors. Uniquely shared by Patr-AL, HLA-A*0201, and other members of the A02 supertype are the absence of serine at position 9 in the B pocket and the presence of tyrosine at position 116 in the F pocket. Distinguishing Patr-AL from HLA-A*02 is an unusually electropositive upper face on the α2 helix. Stimulating PBMC from Patr-AL- chimpanzees with B cells expressing Patr-AL, produced potent alloreactive CD8 T cells with specificity for Patr-AL and no crossreactivity toward other MHC class I, including HLA-A*02. PBMC from Patr-AL+ chimpanzees are tolerant of Patr-AL.
doi:10.4049/jimmunol.1002990
PMCID: PMC3124313  PMID: 21209280
5.  Minority Variants Associated with Transmitted and Acquired HIV-1 Non-Nucleoside RT Inhibitor (NNRTI) Resistance: Implications for the Use of Second Generation NNRTIs 
Objectives
K103N, the most common nonnucleoside (NNRTI)-resistance mutation in patients with transmitted resistance and in patients receiving a failing NNRTI-containing regimen, is fully susceptible to the new NNRTI etravirine. Therefore, we sought to determine how often NNRTI-resistance mutations other than K103N occur as minority variants in plasma samples for which standard genotypic resistance testing (SGRT) detects K103N alone.
Methods
We performed ultradeep pyrosequencing (UDPS; 454 Life Sciences a Roche Company) of plasma virus samples from 13 treatment-naïve and 20 NNRTI-experienced patients in whom SGRT revealed K103N but no other major NNRTI-resistance mutations.
Results
Samples from 0 of 13 treatment-naïve patients vs 7 of 20 patients failing an NNRTI-containing regimen had minority variants with major etravirine-associated NNRTI resistance mutations (p=0.03, Fishers Exact Test): Y181C (7.0%), Y181C (3.6%) + G190A (3.2%), L100I (14%), L100I (32%) + 190A (5.4%), K101E (3.8%) + G190A (4.9%), K101E (4.0%) + G190S (4.8%), and G190S (3.1%).
Conclusion
In treatment-naïve patients, UDPS did not detect additional major NNRTI-resistance mutations suggesting that etravirine may be effective in patients with transmitted K103N. In NNRTI-experienced patients, UDPS often detected additional major NNRTI-resistance mutations suggesting that etravirine may not be fully active in patients with acquired K103N.
doi:10.1097/QAI.0b013e3181bca669
PMCID: PMC2809083  PMID: 19734799
Reverse transcriptase (RT); Nonnucleoside RT inhibitors; Drug resistance mutations; Etravirine; Pyrosequencing; Quasispecies
6.  Pyrosequencing of the DNA gyrase gene in Neisseria species: effective indicator of ciprofloxacin resistance in Neisseria gonorrhoeae 
The quinolone resistance determining region (QRDR) of the gyrA gene in ciprofloxacin-susceptible strains (n=53) and strains of Neisseria spp. with reduced susceptibility (n=70) was determined by the pyrosequencing method. Results showed that the QRDR of the gyrA gene is an effective molecular indicator of resistance to ciprofloxacin in Neisseria gonorrhoeae, and presumably in Neisseria meningitidis, but not in all other Neisseria spp. This sequence was not unique for N. gonorrhoeae and seems unsuitable for species verification of N. gonorrhoeae. However, whether it is also possible to use this region for verification depends on the specificity of the primary screening method used.
doi:10.1111/j.1600-0463.2006.apm_495.x
PMCID: PMC2953271  PMID: 17207083
Neisseria gonorrhoeae; Neisseria species; ciprofloxacin; gyrA; species verification
7.  Sentinel-base DNA genotyping using multiple sequencing primers for high-risk human papillomaviruses 
Molecular and cellular probes  2006;20(3-4):230-238.
Despite the various technologies in place for genotyping human papillomaviruses (HPV), clinical use and clinical research demand a method that is fast, more reliable and cost-effective. The technology described here represents a breakthrough development in that direction. By combining the method of multiple sequencing primers with DNA sequencing, we have developed a rapid assay for genotyping HPV that relies on the identification of a single, type-specific ‘sentinel’ base. As described here, the prototype assay has been developed to recognize the 12 most high-risk HPV types (HPV-16, 18, 31, 33, 35, 39, 45, 51, 52, 56, 58 and 59) and is capable of recognizing and simultaneously genotyping multiple HPV co-infections. By providing sequence information on multiple HPV infections, this method eliminates the need for labor- and cost-intensive PCR cloning. These proof-of-concept studies establish the assay to be accurate, reliable, rapid, flexible, and cost-effective, providing evidence of the feasibility this technique for use in clinical settings.
doi:10.1016/j.mcp.2006.01.002
PMCID: PMC2932960  PMID: 16516439
Human papillomaviruses (HPV); DNA sequencing; Multiple infections; Multiple sequencing primers; Sentinel-base DNA sequencing; Pyrosequencing technology
8.  Large-scale Pyrosequencing of synthetic DNA: A comparison with results from Sanger dideoxy sequencing 
Electrophoresis  2006;27(15):3042-3047.
Pyrosequencing is a relatively recent method for sequencing short stretches of DNA. Because both Pyrosequencing and Sanger dideoxy sequencing were recently used to characterize and validate DNA molecular barcodes in a large yeast gene-deletion project, a meta-analysis of those data allow an excellent and timely opportunity for evaluating Pyrosequencing against the current gold standard, Sanger dideoxy sequencing. Starting with yeast genomic DNA, parallel PCR amplification methods were used to prepared 4747 short barcode-containing constructs from 6000 Saccharomyces cerevisiae gene-deletion strains. Pyrosequencing was optimized for average read lengths of 25–30 bases, which included in each case a 20-mer barcode sequence. Results were compared with sequence data obtained by the standard Sanger dideoxy chain termination method. In most cases, sequences obtained by Pyrosequencing and Sanger dideoxy sequencing were of comparable accuracy, and the overall rate of failure was similar. The DNA in the barcodes is derived from synthetic oligonucleotide sequences that were inserted into yeast-deletion-strain genomic DNA by homologous recombination and represents the most significant amount of DNA from a synthetic source that has been sequenced to date. Although more automation and quality control measures are needed, Pyrosequencing was shown to be a fast and convenient method for determining short stretches of DNA sequence.
doi:10.1002/elps.200500834
PMCID: PMC2932963  PMID: 16800029
DNA sequencing; Pyrosequencing technology; Sanger dideoxy sequencing; Synthetic oligonucleotides; Yeast deletion strains
9.  Does HPV-status 6–12 months after treatment of high grade dysplasia in the uterine cervix predict long term recurrence? 
Background
Women once treated for high grade cervical dysplasia have a high long term risk for developing new dysplasia or cancer.
Objectives
To investigate if human papilloma virus (HPV)-negativity after treatment of cervical dysplasia reduces the need for frequent long term follow up.
Design
Case/control study based on archival smears.
Methods
Women with cervical intraepithelial neoplasi (CIN)2–3, treated for dysplasia and with recurrence of CIN2+ more than 2 years after treatment were compared with controls without recurrence, matched for age and date of treatment. High risk-HPV-DNA were analysed with PCR from two archival smears per woman. Mean follow up time was 14.6 years.
Results
24% (45/189) of cases and 11% (43/378) of controls were HPV-positive in any of two smears. Odds ratio (OR) = 2.5 (1.6–3.8).
Conclusion
HPV-status 6–12 months after treatment of high grade dysplasia is of limited value for the design of long term follow up.
doi:10.1016/j.ejca.2007.05.016
PMCID: PMC2933034  PMID: 17614272
HPV; Cervical intraepithelial neoplasia; Follow-up; Recurrence; Treatment; Case–control studies
10.  Methodological improvements of pyrosequencing technology 
Journal of biotechnology  2006;124(3):504-511.
Pyrosequencing technology is a rather novel DNA sequencing method based on the sequencing-by-synthesis principle. This bioluminometric, real-time DNA sequencing technique employs a cascade of four enzymatic reactions producing sequence peak signals. The method has been proven highly suitable for single nucleotide polymorphism analysis and sequencing of short stretches of DNA. Although the pyrosequencing procedure is relatively straightforward, users may face challenges due to varying parameters in PCR and sequencing primer design, sample preparation and nucleotide dispensation; such challenges are labor and cost intensive. In this study, these issues have been addressed to increase signal quality and assure sequence accuracy.
doi:10.1016/j.jbiotec.2006.01.025
PMCID: PMC2933062  PMID: 16530870
DNA sequencing; Primer-dimers; Pyrosequencing technology; Sample preparation; Single-strand binding protein
11.  Nucleic Acid Template and the Risk of a PCR-Induced HIV-1 Drug Resistance Mutation 
PLoS ONE  2010;5(6):e10992.
Background
The HIV-1 nucleoside RT inhibitor (NRTI)-resistance mutation, K65R confers intermediate to high-level resistance to the NRTIs abacavir, didanosine, emtricitabine, lamivudine, and tenofovir; and low-level resistance to stavudine. Several lines of evidence suggest that K65R is more common in HIV-1 subtype C than subtype B viruses.
Methods and Findings
We performed ultra-deep pyrosequencing (UDPS) and clonal dideoxynucleotide sequencing of plasma virus samples to assess the prevalence of minority K65R variants in subtype B and C viruses from untreated individuals. Although UDPS of plasma samples from 18 subtype C and 27 subtype B viruses showed that a higher proportion of subtype C viruses contain K65R (1.04% vs. 0.25%; p<0.001), limiting dilution clonal sequencing failed to corroborate its presence in two of the samples in which K65R was present in >1.5% of UDPS reads. We therefore performed UDPS on clones and site-directed mutants containing subtype B- and C-specific patterns of silent mutations in the conserved KKK motif encompassing RT codons 64 to 66 and found that subtype-specific nucleotide differences were responsible for increased PCR-induced K65R mutation in subtype C viruses.
Conclusions
This study shows that the RT KKK nucleotide template in subtype C viruses can lead to the spurious detection of K65R by highly sensitive PCR-dependent sequencing techniques. However, the study is also consistent with the subtype C nucleotide template being inherently responsible for increased polymerization-induced K65R mutations in vivo.
doi:10.1371/journal.pone.0010992
PMCID: PMC2881873  PMID: 20539818
12.  Six RNA Viruses and Forty-One Hosts: Viral Small RNAs and Modulation of Small RNA Repertoires in Vertebrate and Invertebrate Systems 
PLoS Pathogens  2010;6(2):e1000764.
We have used multiplexed high-throughput sequencing to characterize changes in small RNA populations that occur during viral infection in animal cells. Small RNA-based mechanisms such as RNA interference (RNAi) have been shown in plant and invertebrate systems to play a key role in host responses to viral infection. Although homologs of the key RNAi effector pathways are present in mammalian cells, and can launch an RNAi-mediated degradation of experimentally targeted mRNAs, any role for such responses in mammalian host-virus interactions remains to be characterized. Six different viruses were examined in 41 experimentally susceptible and resistant host systems. We identified virus-derived small RNAs (vsRNAs) from all six viruses, with total abundance varying from “vanishingly rare” (less than 0.1% of cellular small RNA) to highly abundant (comparable to abundant micro-RNAs “miRNAs”). In addition to the appearance of vsRNAs during infection, we saw a number of specific changes in host miRNA profiles. For several infection models investigated in more detail, the RNAi and Interferon pathways modulated the abundance of vsRNAs. We also found evidence for populations of vsRNAs that exist as duplexed siRNAs with zero to three nucleotide 3′ overhangs. Using populations of cells carrying a Hepatitis C replicon, we observed strand-selective loading of siRNAs onto Argonaute complexes. These experiments define vsRNAs as one possible component of the interplay between animal viruses and their hosts.
Author Summary
Short RNAs derived from invading viruses with RNA genomes are important components of antiviral immunity in plants, worms and flies. The regulated generation of these short RNAs, and their engagement by the immune apparatus, is essential for inhibiting viral growth in these organisms. Mammals have the necessary protein components to generate these viral-derived short RNAs (“vsRNAs”), raising the question of whether vsRNAs in mammals are a general feature of infections with RNA viruses. Our work with Hepatitis C, Polio, Dengue, Vesicular Stomatitis, and West Nile viruses in a broad host repertoire demonstrates the generality of RNA virus-derived vsRNA production, and the ability of the cellular short RNA apparatus to engage these vsRNAs in mammalian cells. Detailed analyses of vsRNA and host-derived short RNA populations demonstrate both common and virus-specific features of the interplay between viral infection and short RNA populations. The vsRNA populations described in this work represent a novel dimension in both viral pathogenesis and host response.
doi:10.1371/journal.ppat.1000764
PMCID: PMC2820531  PMID: 20169186
13.  Detection of gyrA mutations associated with ciprofloxacin resistance in Neisseria gonorrhoeae by rapid and reliable pre-programmed short DNA sequencing 
Quinolone resistance is rapidly increasing in Neisseria gonorrhoeae and is posing a significant public health threat that requires constant surveillance. A rapid and reliable mutation detection assay has been developed. The assay is based on pre-programmed short DNA sequencing and is designed to detect point mutations in the gyrA gene that are highly related to ciprofloxacin resistance, i.e. in codons 91 and 95. By developing an assay based on pyrosequencing and exploiting the pre-programmed nucleotide dispensation capability of this technology, the sequence comprising the mutations will be analysed and promptly reveal whether the N. gonorrhoeae pathogen carries resistance to ciprofloxacin. A panel of 40 N. gonorrhoeae clinical isolates, of which 27 phenotypically displayed decreased susceptibility or resistance to ciprofloxacin, was used in the present study. All point mutations in the short stretch of the N. gonorrhoeae gyrA gene were easily discriminated, and the genotypic results obtained by pre-programmed sequencing were mainly in agreement with the phenotypically identified decreased susceptibility or resistance to ciprofloxacin. The new method used in the present study has the potential for rapid and reliable identification of known as well as previously unknown drug resistance mutations.
doi:10.1016/j.ijantimicag.2005.08.017
PMCID: PMC2768773  PMID: 16274961
DNA sequencing; Ciprofloxacin resistance; Neisseria gonorrhoeae; Pre-programmed DNA sequencing; Pyrosequencing technology
14.  Molecular characterization of Neisseria gonorrhoeae identifies transmission and resistance of one ciprofloxacin-resistant strain 
A highly discriminative and objective genetic characterization of N. gonorrhoeae, which increases our knowledge of strain populations in different geographic areas, is crucial for the development of improved control measures. In the present study, conventional phenotypic characterization and genetic characterization by means of pulsed-field gel electrophoresis (PFGE), sequencing of the entire porB gene, N. gonorrhoeae multiantigen sequence typing (NG-MAST), and pyrosequencing of a quinolone resistance determining region (QRDR) of the gyrA gene of Swedish ciprofloxacin-resistant N. gonorrhoeae serovar IB-10 isolates (n=45) were performed. The genetic characterization identified one widely spread ciprofloxacin-resistant N. gonorrhoeae ST147 strain. In addition, isolates with slightly different genetic characteristics, which presumably reflect the ongoing evolution only, were also identified. All the isolates contained single nucleotide polymorphisms in QRDR of the gyrA gene that are highly correlated with ciprofloxacin resistance. Consequently, comprehensive characterization identified the first confirmed large domestic transmission, mainly among young heterosexuals, of one ciprofloxacin-resistant N. gonorrhoeae strain in Swedish society during 2002–2003. In conclusion, a precise, i.e. genetic, characterization for identification of individual strains is a very valuable support to the crucial active surveillance of the epidemiological characteristics and the antibiotic susceptibility of N. gonorrhoeae in the effective treatment of gonorrhoea.
doi:10.1111/j.1600-0463.2007.apm_487.x
PMCID: PMC2769514  PMID: 17367469
Neisseria gonorrhoeae; ciprofloxacin resistance; molecular epidemiology; porB gene; NG-MAST
15.  Minority Human Immunodeficiency Virus Type 1 Variants in Antiretroviral-Naive Persons with Reverse Transcriptase Codon 215 Revertant Mutations▿ †  
Journal of Virology  2008;82(21):10747-10755.
T215 revertant mutations such as T215C/D/E/S that evolve from the nucleoside reverse transcriptase (RT) inhibitor mutations T215Y/F have been found in about 3% of human immunodeficiency virus type 1 (HIV-1) isolates from newly diagnosed HIV-1-infected persons. We used a newly developed sequencing method—ultradeep pyrosequencing (UDPS; 454 Life Sciences)—to determine the frequency with which T215Y/F or other RT inhibitor resistance mutations could be detected as minority variants in samples from untreated persons that contain T215 revertants (“revertant” samples) compared with samples from untreated persons that lack such revertants (“control” samples). Among the 22 revertant and 29 control samples, UDPS detected a mean of 3.8 and 4.8 additional RT amino acid mutations, respectively. In 6 of 22 (27%) revertant samples and in 4 of 29 control samples (14%; P = 0.4), UDPS detected one or more RT inhibitor resistance mutations. T215Y or T215F was not detected in any of the revertant or control samples; however, 4 of 22 revertant samples had one or more T215 revertants that were detected by UDPS but not by direct PCR sequencing. The failure to detect viruses with T215Y/F in the 22 revertant samples in this study may result from the overwhelming replacement of transmitted T215Y variants by the more fit T215 revertants or from the primary transmission of a T215 revertant in a subset of persons with T215 revertants.
doi:10.1128/JVI.01827-07
PMCID: PMC2573178  PMID: 18715933
16.  High Throughput Automated Allele Frequency Estimation by Pyrosequencing 
PLoS ONE  2008;3(7):e2693.
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.
doi:10.1371/journal.pone.0002693
PMCID: PMC2442187  PMID: 18628978
17.  Viral Population Estimation Using Pyrosequencing 
PLoS Computational Biology  2008;4(5):e1000074.
The diversity of virus populations within single infected hosts presents a major difficulty for the natural immune response as well as for vaccine design and antiviral drug therapy. Recently developed pyrophosphate-based sequencing technologies (pyrosequencing) can be used for quantifying this diversity by ultra-deep sequencing of virus samples. We present computational methods for the analysis of such sequence data and apply these techniques to pyrosequencing data obtained from HIV populations within patients harboring drug-resistant virus strains. Our main result is the estimation of the population structure of the sample from the pyrosequencing reads. This inference is based on a statistical approach to error correction, followed by a combinatorial algorithm for constructing a minimal set of haplotypes that explain the data. Using this set of explaining haplotypes, we apply a statistical model to infer the frequencies of the haplotypes in the population via an expectation–maximization (EM) algorithm. We demonstrate that pyrosequencing reads allow for effective population reconstruction by extensive simulations and by comparison to 165 sequences obtained directly from clonal sequencing of four independent, diverse HIV populations. Thus, pyrosequencing can be used for cost-effective estimation of the structure of virus populations, promising new insights into viral evolutionary dynamics and disease control strategies.
Author Summary
The genetic diversity of viral populations is important for biomedical problems such as disease progression, vaccine design, and drug resistance, yet it is not generally well understood. In this paper, we use pyrosequencing, a novel DNA sequencing technique, to reconstruct viral populations. Pyrosequencing produces DNA sequences, called reads, in numbers much greater than standard DNA sequencing techniques. However, these reads are substantially shorter and more error-prone than those obtained from standard sequencing techniques. Therefore, pyrosequencing data requires new methods of analysis. Here, we develop mathematical and statistical tools for reconstructing viral populations using pyrosequencing. To this end, we show how to correct errors in the reads and assemble them into the different viral strains present in the population. We apply these methods to HIV-1 populations from drug-resistant patients and show that our techniques produce results quite close to accepted techniques at a lower cost and potentially higher resolution.
doi:10.1371/journal.pcbi.1000074
PMCID: PMC2323617  PMID: 18437230
18.  A pyrosequencing-tailored nucleotide barcode design unveils opportunities for large-scale sample multiplexing 
Nucleic Acids Research  2007;35(19):e130.
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%.
doi:10.1093/nar/gkm760
PMCID: PMC2095802  PMID: 17932070
19.  Viral and microbial genotyping by a combination of multiplex competitive hybridization and specific extension followed by hybridization to generic tag arrays 
Nucleic Acids Research  2003;31(22):e146.
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.
doi:10.1093/nar/gng147
PMCID: PMC275583  PMID: 14602939
20.  Genotyping by apyrase-mediated allele-specific extension 
Nucleic Acids Research  2001;29(24):e121.
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.
PMCID: PMC97629  PMID: 11812858

Results 1-20 (20)