Sequencing of a single-cell genome requires DNA amplification, a process prone to introducing bias and errors into the amplified genome. Here we introduce a novel multiple displacement amplification (MDA) method based on the unique DNA primase features of Thermus thermophilus (Tth) PrimPol. TthPrimPol displays a potent primase activity preferring dNTPs as substrates unlike conventional primases. A combination of TthPrimPol’s unique ability to synthesize DNA primers with the highly processive Phi29 DNA polymerase (Φ29DNApol) enables near-complete whole genome amplification from single cells. This novel method demonstrates superior breadth and evenness of genome coverage, high reproducibility, excellent single-nucleotide variant (SNV) detection rates with low allelic dropout (ADO) and low chimera formation as exemplified by sequencing HEK293 cells. Moreover, copy number variant (CNV) calling yields superior results compared with random primer-based MDA methods. The advantages of this method, which we named TruePrime, promise to facilitate and improve single-cell genomic analysis.

Significance: The complexity of the human memory B-lymphocyte compartment is a key component to depict and understand adaptive immunity. Despite numerous prior investigations, the generation of certain memory B-cell subsets, the dependency on T-cell help, and the composition, size, and diversity of clonal expansions are either poorly understood or debated. Here we provide an extensive and tightly controlled immunoglobulin heavy chain variable (IGHV) gene repertoire analysis of four main human memory B-cell subpopulations, revealing that an ordered diversification in germinal centers determines a highly versatile memory B-cell compartment in humans with surprisingly many very large B-cell clones.

High throughput sequencing (HTSeq) of small ribosomal subunit amplicons has the potential for a comprehensive characterization of microbial community compositions, down to rare species. However, the error-prone nature of the multi-step experimental process requires that the resulting raw sequences are subjected to quality control procedures. These procedures often involve an abundance cutoff for rare sequences or clustering of sequences, both of which limit genetic resolution. Here we propose a simple experimental protocol that retains the high genetic resolution granted by HTSeq methods while effectively removing many low abundance sequences that are likely due to PCR and sequencing errors. According to this protocol, we split samples and submit both halves to independent PCR and sequencing runs. The resulting sequence data is graphically and quantitatively characterized by the discordance between the two experimental branches, allowing for a quick identification of problematic samples. Further, we discard sequences that are not found in both branches (“AmpliconDuo filter”). We show that the majority of sequences removed in this way, mostly low abundance but also some higher abundance sequences, show features expected from random modifications of true sequences as introduced by PCR and sequencing errors. On the other hand, the filter retains many low abundance sequences observed in both branches and thus provides a more reliable census of the rare biosphere. We find that the AmpliconDuo filter increases biological resolution as it increases apparent community similarity between biologically similar communities, while it does not affect apparent community similarities between biologically dissimilar communities. The filter does not distort overall apparent community compositions. Finally, we quantitatively explain the effect of the AmpliconDuo filter by a simple mathematical model.

Background & Aims Hepatitis D virus (HDV) superinfection in patients with hepatitis B virus (HBV) is associated with rapid progression to liver cirrhosis and hepatocellular carcinoma. Treatment options are limited, and no vaccine is available. Although HDV-specific CD8⁺ T cells are thought to control the virus, little is known about which HDV epitopes are targeted by virus-specific CD8⁺ T cells or why these cells ultimately fail to control the infection. We aimed to define how HDV escapes the CD8⁺ T-cell–mediated response. Methods We collected plasma and DNA samples from 104 patients with chronic HDV and HBV infection at medical centers in Europe and the Middle East, sequenced HDV, typed human leukocyte antigen (HLA) class I alleles from patients, and searched for polymorphisms in HDV RNA associated with specific HLA class I alleles. We predicted epitopes in HDV that would be recognized by CD8⁺ T cells and corresponded with the identified virus polymorphisms in patients with resolved (n = 12) or chronic (n = 13) HDV infection. Results We identified 21 polymorphisms in HDV that were significantly associated with specific HLA class I alleles (P < .005). Five of these polymorphisms were found to correspond to epitopes in HDV that are recognized by CD8⁺ T cells; we confirmed that CD8⁺ T cells in culture targeted these HDV epitopes. HDV variant peptides were only partially cross-recognized by CD8⁺ T cells isolated from patients, indicating that the virus had escaped detection by these cells. These newly identified HDV epitopes were restricted by relatively infrequent HLA class I alleles, and they bound most frequently to HLA-B. In contrast, frequent HLA class I alleles were not associated with HDV sequence polymorphisms. Conclusions We analyzed sequences of HDV RNA and HLA class I alleles that present epitope peptides to CD8⁺ T cells in patients with persistent HDV infection. We identified polymorphisms in the HDV proteome that associate with HLA class I alleles. Some variant peptides in epitopes from HDV were only partially recognized by CD8⁺ T cells isolated from patients; these could be mutations that allow HDV to escape the immune response, resulting in persistent infection. HDV escape from the immune response was associated with uncommon HLA class I alleles, indicating that HDV evolves, at the population level, to evade recognition by common HLA class I alleles.

Activation of hepatitis B virus (HBV)–specific CD8 T cells by therapeutic vaccination may promote sustained control of viral replication by clearance of covalently closed circular DNA from infected hepatocytes. However, little is known about the exact targets of the CD8 T‐cell response and whether HBV reproducibly evades CD8 T‐cell immune pressure by mutation. The aim of this study was to address if HBV reproducibly selects substitutions in CD8 T‐cell epitopes that functionally act as immune escape mutations. The HBV core gene was amplified and sequenced from 148 patients with chronic HBV infection, and the human leukocyte antigen (HLA) class I genotype (A and B loci) was determined. Residues under selection pressure in the presence of particular HLA class I alleles were identified by a statistical approach utilizing the novel analysis package SeqFeatR. With this approach we identified nine residues in HBV core under selection pressure in the presence of 10 different HLA class I alleles. Additional immunological experiments confirmed that seven of the residues were located inside epitopes targeted by patients with chronic HBV infection carrying the relevant HLA class I allele. Consistent with viral escape, the selected substitutions reproducibly impaired recognition by HBV‐specific CD8 T cells. Conclusion: Viral sequence analysis allows identification of HLA class I–restricted epitopes under reproducible selection pressure in HBV core; the possibility of viral escape from CD8 T‐cell immune pressure needs attention in the context of therapeutic vaccination against HBV. (Hepatology 2015;62:47‐56)