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.

The detection of mutant spectra within a population of microorganisms is critical for the management of drug-resistant infections. We performed ultra-deep pyrosequencing to detect minor sequence variants in HIV-1 protease and reverse transcriptase (RT) genes from clinical plasma samples. We estimated empirical error rates from four HIV-1 plasmid clones and used them to develop a statistical approach to distinguish authentic minor variants from sequencing errors in eight clinical samples. Ultra-deep pyrosequencing detected an average of 58 variants per sample compared with an average of eight variants per sample detected by conventional direct-PCR dideoxynucleotide sequencing. In the clinical sample with the largest number of minor sequence variants, all 60 variants present in Ն3% of genomes and 20 of 35 variants present in <3% of genomes were confirmed by limiting dilution sequencing. With appropriate analysis, ultra-deep pyrosequencing is a promising method for characterizing genetic diversity and detecting minor yet clinically relevant variants in biological samples with complex genetic populations.[Supplemental material is available online at www.genome.org. The raw data from this study are available online at http://dbpartners.stanford.edu/454/pub.] Dideoxynucleotide (Sanger) sequencing of non-clonal PCR products (direct PCR sequencing) of plasma viral cDNA is widely used to detect more than 50 drug-resistance mutations in the molecular targets of HIV-1 therapy-reverse transcriptase (RT) and protease-in clinical settings (US Department of Health and Human Services Panel on Clinical Practices for Treatment of HIV Infection 2006). A major limitation of direct PCR sequencing, however, is its inability to detect low proportions of drug-resistant variants in the heterogeneous virus population existing in a patient's plasma sample (Palmer et al. 2005). Several studies have shown that minor drug-resistant variants that are not detected by population-based sequencing are clinically relevant in that they are often responsible for the virological failure of a new antiretroviral treatment regimen (Jourdain et al. 2004;Kapoor et al. 2004;Lecossier et al. 2005;Palmer et al. 2006b). Multiple approaches have been developed to detect minor HIV-1 variants in research settings; however, no single approach has proved useful for clinical settings.The 454 Life Sciences GS20 sequencing platform allows massively parallel picoliter-scale amplification and pyrosequencing of individual DNA molecules (Margulies et al. 2005). Simons and colleagues described two cases in which ultra-deep pyrosequencing detected minority variant drug-resistance mutations in a previously treated patient in whom mutations were no longer detectable by standard direct PCR sequencing (Simons et al. 2005). Tsibris and colleagues demonstrated that ultra-deep pyrosequencing could accurately quantify a mixture of three HIV-1 envelope variants pooled in defined proportions of 89%, 10%, and 1% (Tsibris et al. 2006). Here, we systematically investigate the potential...

Developing T cells face a series of cell fate choices in the thymus and in the periphery. The role of the individual T cell receptor (TCR) in determining decisions of cell fate remains unresolved. The stochastic/selection model postulates that the initial fate of the cell is independent of TCR specificity, with survival dependent on additional TCR/coreceptor "rescue" signals. The "instructive" model holds that cell fate is initiated by the interaction of the TCR with a cognate peptide-MHC complex. T cells are then segregated on the basis of TCR specificity with the aid of critical coreceptors and signal modulators [Chan S, Correia-Neves M, Benoist C, Mathis (1998) Immunol Rev 165: 195-207]. The former would predict a random representation of individual TCR across divergent T cell lineages whereas the latter would predict minimal overlap between divergent T cell subsets. To address this issue, we have used highthroughput sequencing to evaluate the TCR distribution among key T cell developmental and effector subsets from a single donor. We found numerous examples of individual subsets sharing identical TCR sequence, supporting a model of a stochastic process of cell fate determination coupled with dynamic patterns of clonal expansion of T cells bearing the same TCR sequence among both CD4 + and CD8+ populations.F ollowing production of their T cell receptors (TCRs), T cells experience several developing stages. An encounter with a cognate peptide-MHC complex can induce naïve T (Tn) cells expressing the CD45RA isomer to begin to express CD45RO. Cells expressing both isomers are considered transitional in nature (Tt), thus cells identified on the basis of CD45RA expression alone include Tn and Tt and can thus be referred to as Tn+t. Cells expressing only CD45RO have passed into the memory (Tm) compartment, where they can lay quiescent awaiting repeat stimulation by the same or similar peptide-MHC complexes. Activated T cells (Ta) driven to effector function lose expression of both CD45RA and RO and express CD69. During different developing stages, T cells also face a series of cell fate choices: CD4 + CD8+ cells commit to either the CD4 + helper (Th) or CD8+ cytotoxic (Tc) lineages, a choice closely associated with binding to MHC class II or class I peptide complexes, respectively. Subsequently, CD4 + T cells can develop into regulatory (Tr) CD25+ cells, or into CD25−CD294− Th1 (IFN-γ producing) or CD25−CD294+ Th2 (IL-4 producing) effector subsets. Other choices are also available (1, 2).Although it is generally accepted that the TCR expressed by the developing T lineage cell will determine the response to a specific peptide-MHC complex, the role of the individual TCR in determining decisions of cell fate remains unresolved. To address these issues, we have coupled high-throughput sequencing techniques (3, 4) to high volume antibody covered superparamagnetic polystyrene bead isolation of defined T cell subsets with semiquantitative PCR amplification of the complementarity determining region 3 regions (CDR3) from mR...

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.

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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 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.

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.