Single T Cell Sequencing Demonstrates the Functional Role of αß TCR Pairing in Cell Lineage and Antigen Specificity.

Although structural studies of individual T cell receptors (TCRs) have revealed important roles for both the α and ß chain in directing MHC and antigen recognition, repertoire-level immunogenomic analyses have historically examined the ß chain alone. To determine the amount of useful information about TCR repertoire function encoded within αß pairings, we analyzed paired TCR sequences from nearly 100,000 unique CD4+ and CD8+ T cells captured using two different high-throughput, single-cell sequencing approaches. Our results demonstrate little overlap in the healthy CD4+ and CD8+ repertoires, with shared TCR sequences possessing significantly shorter CDR3 sequences corresponding to higher generation probabilities. We further utilized tools from information theory and machine learning to show that while α and ß chains are only weakly associated with lineage, αß pairings appear to synergistically drive TCR-MHC interactions. Vαß gene pairings were found to be the TCR feature most informative of T cell lineage, supporting the existence of germline-encoded paired αß TCR-MHC interaction motifs. Finally, annotating our TCR pairs using a database of sequences with known antigen specificities, we demonstrate that approximately a third of the T cells possess α and ß chains that each recognize different known antigens, suggesting that αß pairing is critical for the accurate inference of repertoire functionality. Together, these findings provide biological insight into the functional implications of αß pairing and highlight the utility of single-cell sequencing in immunogenomics.

Corrigendum: Engineering and optimising deaminase fusions for genome editing.

This corrects the article DOI: 10.1038/ncomms13330.

Hierarchical Clustering Can Identify B Cell Clones with High Confidence in Ig Repertoire Sequencing Data.

Adaptive immunity is driven by the expansion, somatic hypermutation, and selection of B cell clones. Each clone is the progeny of a single B cell responding to Ag, with diversified Ig receptors. These receptors can now be profiled on a large scale by next-generation sequencing. Such data provide a window into the microevolutionary dynamics that drive successful immune responses and the dysregulation that occurs with aging or disease. Clonal relationships are not directly measured, but they must be computationally inferred from these sequencing data. Although several hierarchical clustering-based methods have been proposed, they vary in distance and linkage methods and have not yet been rigorously compared. In this study, we use a combination of human experimental and simulated data to characterize the performance of hierarchical clustering-based methods for partitioning sequences into clones. We find that single linkage clustering has high performance, with specificity, sensitivity, and positive predictive value all >99%, whereas other linkages result in a significant loss of sensitivity. Surprisingly, distance metrics that incorporate the biases of somatic hypermutation do not outperform simple Hamming distance. Although errors were more likely in sequences with short junctions, using the entire dataset to choose a single distance threshold for clustering is near optimal. Our results suggest that hierarchical clustering using single linkage with Hamming distance identifies clones with high confidence and provides a fully automated method for clonal grouping. The performance estimates we develop provide important context to interpret clonal analysis of repertoire sequencing data and allow for rigorous testing of other clonal grouping algorithms.

Engineering and optimising deaminase fusions for genome editing.

Precise editing is essential for biomedical research and gene therapy. Yet, homology-directed genome modification is limited by the requirements for genomic lesions, homology donors and the endogenous DNA repair machinery. Here we engineered programmable cytidine deaminases and test if we could introduce site-specific cytidine to thymidine transitions in the absence of targeted genomic lesions. Our programmable deaminases effectively convert specific cytidines to thymidines with 13% efficiency in Escherichia coli and 2.5% in human cells. However, off-target deaminations were detected more than 150 bp away from the target site. Moreover, whole genome sequencing revealed that edited bacterial cells did not harbour chromosomal abnormalities but demonstrated elevated global cytidine deamination at deaminase intrinsic binding sites. Therefore programmable deaminases represent a promising genome editing tool in prokaryotes and eukaryotes. Future engineering is required to overcome the processivity and the intrinsic DNA binding affinity of deaminases for safer therapeutic applications.

A Model of Somatic Hypermutation Targeting in Mice Based on High-Throughput Ig Sequencing Data.

Analyses of somatic hypermutation (SHM) patterns in B cell Ig sequences have important basic science and clinical applications, but they are often confounded by the intrinsic biases of SHM targeting on specific DNA motifs (i.e., hot and cold spots). Modeling these biases has been hindered by the difficulty in identifying mutated Ig sequences in vivo in the absence of selection pressures, which skew the observed mutation patterns. To generate a large number of unselected mutations, we immunized B1-8 H chain transgenic mice with nitrophenyl to stimulate nitrophenyl-specific λ+ germinal center B cells and sequenced the unexpressed κ L chains using next-generation methods. Most of these κ sequences had out-of-frame junctions and were presumably uninfluenced by selection. Despite being nonfunctionally rearranged, they were targeted by SHM and displayed a higher mutation frequency than functional sequences. We used 39,173 mutations to construct a quantitative SHM targeting model. The model showed targeting biases that were consistent with classic hot and cold spots, yet revealed additional highly mutable motifs. We observed comparable targeting for functional and nonfunctional sequences, suggesting similar biological processes operate at both loci. However, we observed species- and chain-specific targeting patterns, demonstrating the need for multiple SHM targeting models. Interestingly, the targeting of C/G bases and the frequency of transition mutations at C/G bases was higher in mice compared with humans, suggesting lower levels of DNA repair activity in mice. Our models of SHM targeting provide insights into the SHM process and support future analyses of mutation patterns.

Genetic Evidence of Human Adaptation to a Cooked Diet.

Humans have been argued to be biologically adapted to a cooked diet, but this hypothesis has not been tested at the molecular level. Here, we combine controlled feeding experiments in mice with comparative primate genomics to show that consumption of a cooked diet influences gene expression and that affected genes bear signals of positive selection in the human lineage. Liver gene expression profiles in mice fed standardized diets of meat or tuber were affected by food type and cooking, but not by caloric intake or consumer energy balance. Genes affected by cooking were highly correlated with genes known to be differentially expressed in liver between humans and other primates, and more genes in this overlap set show signals of positive selection in humans than would be expected by chance. Sequence changes in the genes under selection appear before the split between modern humans and two archaic human groups, Neandertals and Denisovans, supporting the idea that human adaptation to a cooked diet had begun by at least 275,000 years ago.

Massively parallel sequencing of single cells by epicPCR links functional genes with phylogenetic markers.

Many microbial communities are characterized by high genetic diversity. 16S ribosomal RNA sequencing can determine community members, and metagenomics can determine the functional diversity, but resolving the functional role of individual cells in high throughput remains an unsolved challenge. Here, we describe epicPCR (Emulsion, Paired Isolation and Concatenation PCR), a new technique that links functional genes and phylogenetic markers in uncultured single cells, providing a throughput of hundreds of thousands of cells with costs comparable to one genomic library preparation. We demonstrate the utility of our technique in a natural environment by profiling a sulfate-reducing community in a freshwater lake, revealing both known sulfate reducers and discovering new putative sulfate reducers. Our method is adaptable to any conserved genetic trait and translates genetic associations from diverse microbial samples into a sequencing library that answers targeted ecological questions. Potential applications include identifying functional community members, tracing horizontal gene transfer networks and mapping ecological interactions between microbial cells.

Neutralizing antibodies against West Nile virus identified directly from human B cells by single-cell analysis and next generation sequencing.

West Nile virus (WNV) infection is an emerging mosquito-borne disease that can lead to severe neurological illness and currently has no available treatment or vaccine. Using microengraving, an integrated single-cell analysis method, we analyzed a cohort of subjects infected with WNV - recently infected and post-convalescent subjects - and efficiently identified four novel WNV neutralizing antibodies. We also assessed the humoral response to WNV on a single-cell and repertoire level by integrating next generation sequencing (NGS) into our analysis. The results from single-cell analysis indicate persistence of WNV-specific memory B cells and antibody-secreting cells in post-convalescent subjects. These cells exhibited class-switched antibody isotypes. Furthermore, the results suggest that the antibody response itself does not predict the clinical severity of the disease (asymptomatic or symptomatic). Using the nucleotide coding sequences for WNV-specific antibodies derived from single cells, we revealed the ontogeny of expanded WNV-specific clones in the repertoires of recently infected subjects through NGS and bioinformatic analysis. This analysis also indicated that the humoral response to WNV did not depend on an anamnestic response, due to an unlikely previous exposure to the virus. The innovative and integrative approach presented here to analyze the evolution of neutralizing antibodies from natural infection on a single-cell and repertoire level can also be applied to vaccine studies, and could potentially aid the development of therapeutic antibodies and our basic understanding of other infectious diseases.

Optimization of scarless human stem cell genome editing.

Efficient strategies for precise genome editing in human-induced pluripotent cells (hiPSCs) will enable sophisticated genome engineering for research and clinical purposes. The development of programmable sequence-specific nucleases such as Transcription Activator-Like Effectors Nucleases (TALENs) and Cas9-gRNA allows genetic modifications to be made more efficiently at targeted sites of interest. However, many opportunities remain to optimize these tools and to enlarge their spheres of application. We present several improvements: First, we developed functional re-coded TALEs (reTALEs), which not only enable simple one-pot TALE synthesis but also allow TALE-based applications to be performed using lentiviral vectors. We then compared genome-editing efficiencies in hiPSCs mediated by 15 pairs of reTALENs and Cas9-gRNA targeting CCR5 and optimized ssODN design in conjunction with both methods for introducing specific mutations. We found Cas9-gRNA achieved 7-8× higher non-homologous end joining efficiencies (3%) than reTALENs (0.4%) and moderately superior homology-directed repair efficiencies (1.0 versus 0.6%) when combined with ssODN donors in hiPSCs. Using the optimal design, we demonstrated a streamlined process to generated seamlessly genome corrected hiPSCs within 3 weeks.

A high-coverage genome sequence from an archaic Denisovan individual.

We present a DNA library preparation method that has allowed us to reconstruct a high-coverage (30×) genome sequence of a Denisovan, an extinct relative of Neandertals. The quality of this genome allows a direct estimation of Denisovan heterozygosity indicating that genetic diversity in these archaic hominins was extremely low. It also allows tentative dating of the specimen on the basis of "missing evolution" in its genome, detailed measurements of Denisovan and Neandertal admixture into present-day human populations, and the generation of a near-complete catalog of genetic changes that swept to high frequency in modern humans since their divergence from Denisovans.

Iterative capped assembly: rapid and scalable synthesis of repeat-module DNA such as TAL effectors from individual monomers.

DNA built from modular repeats presents a challenge for gene synthesis. We present a solid surface-based sequential ligation approach, which we refer to as iterative capped assembly (ICA), that adds DNA repeat monomers individually to a growing chain while using hairpin 'capping' oligonucleotides to block incompletely extended chains, greatly increasing the frequency of full-length final products. Applying ICA to a model problem, construction of custom transcription activator-like effector nucleases (TALENs) for genome engineering, we demonstrate efficient synthesis of TALE DNA-binding domains up to 21 monomers long and their ligation into a nuclease-carrying backbone vector all within 3 h. We used ICA to synthesize 20 TALENs of varying DNA target site length and tested their ability to stimulate gene editing by a donor oligonucleotide in human cells. All the TALENS show activity, with the ones >15 monomers long tending to work best. Since ICA builds full-length constructs from individual monomers rather than large exhaustive libraries of pre-fabricated oligomers, it will be trivial to incorporate future modified TALE monomers with improved or expanded function or to synthesize other types of repeat-modular DNA where the diversity of possible monomers makes exhaustive oligomer libraries impractical.

Stable gene targeting in human cells using single-strand oligonucleotides with modified bases.

Recent advances allow multiplexed genome engineering in E. coli, employing easily designed oligonucleotides to edit multiple loci simultaneously. A similar technology in human cells would greatly expedite functional genomics, both by enhancing our ability to test how individual variants such as single nucleotide polymorphisms (SNPs) are related to specific phenotypes, and potentially allowing simultaneous mutation of multiple loci. However, oligo-mediated targeting of human cells is currently limited by low targeting efficiencies and low survival of modified cells. Using a HeLa-based EGFP-rescue reporter system we show that use of modified base analogs can increase targeting efficiency, in part by avoiding the mismatch repair machinery. We investigate the effects of oligonucleotide toxicity and find a strong correlation between the number of phosphorothioate bonds and toxicity. Stably EGFP-corrected cells were generated at a frequency of ~0.05% with an optimized oligonucleotide design combining modified bases and reduced number of phosphorothioate bonds. We provide evidence from comparative RNA-seq analysis suggesting cellular immunity induced by the oligonucleotides might contribute to the low viability of oligo-corrected cells. Further optimization of this method should allow rapid and scalable genome engineering in human cells.

Preparation of next-generation sequencing libraries from damaged DNA.

Next-generation sequencing (NGS) has revolutionized ancient DNA research, especially when combined with high-throughput target enrichment methods. However, attaining high sequencing depth and accuracy from samples often remains problematic due to the damaged state of ancient DNA, in particular the extremely low copy number of ancient DNA and the abundance of uracil residues derived from cytosine deamination that lead to miscoding errors. It is therefore critical to use a highly efficient procedure for conversion of a raw DNA extract into an adaptor-ligated sequencing library, and equally important to reduce errors from uracil residues. We present a protocol for NGS library preparation that allows highly efficient conversion of DNA fragments into an adaptor-ligated form. The protocol incorporates an option to remove the vast majority of uracil miscoding lesions as part of the library preparation process. The procedure requires only two spin column purification steps and no gel purification or bead handling. Starting from an aliquot of DNA extract, a finished, highly amplified library can be generated in 5 h, or under 3 h if uracil removal is not required.

Rapid retrieval of DNA target sequences by primer extension capture.

There is a widespread need for methods to enrich DNA samples for sequences of interest prior to high-throughput sequencing and to reduce the costs associated with a shotgun approach. While useful for targeting megabase-sized regions in a few samples, hybridization capture approaches such as those using microarrays currently involve bulky handling steps, long incubation times, and high per-sample costs. In contrast, the primer extension capture (PEC) method allows direct selection of small genomic regions from DNA sources within 2 h, with low costs for use with parallel samples. PEC promises useful applications in studies such as ancient DNA or forensic sequencing, taxonomic surveying of metagenomic samples, or genomic mapping of repetitive elements.

Genetic history of an archaic hominin group from Denisova Cave in Siberia.

Using DNA extracted from a finger bone found in Denisova Cave in southern Siberia, we have sequenced the genome of an archaic hominin to about 1.9-fold coverage. This individual is from a group that shares a common origin with Neanderthals. This population was not involved in the putative gene flow from Neanderthals into Eurasians; however, the data suggest that it contributed 4-6% of its genetic material to the genomes of present-day Melanesians. We designate this hominin population 'Denisovans' and suggest that it may have been widespread in Asia during the Late Pleistocene epoch. A tooth found in Denisova Cave carries a mitochondrial genome highly similar to that of the finger bone. This tooth shares no derived morphological features with Neanderthals or modern humans, further indicating that Denisovans have an evolutionary history distinct from Neanderthals and modern humans.

Road blocks on paleogenomes--polymerase extension profiling reveals the frequency of blocking lesions in ancient DNA.

Although the last few years have seen great progress in DNA sequence retrieval from fossil specimens, some of the characteristics of ancient DNA remain poorly understood. This is particularly true for blocking lesions, i.e. chemical alterations that cannot be bypassed by DNA polymerases and thus prevent amplification and subsequent sequencing of affected molecules. Some studies have concluded that the vast majority of ancient DNA molecules carry blocking lesions, suggesting that the removal, repair or bypass of blocking lesions might dramatically increase both the time depth and geographical range of specimens available for ancient DNA analysis. However, previous studies used very indirect detection methods that did not provide conclusive estimates on the frequency of blocking lesions in endogenous ancient DNA. We developed a new method, polymerase extension profiling (PEP), that directly reveals occurrences of polymerase stalling on DNA templates. By sequencing thousands of single primer extension products using PEP methodology, we have for the first time directly identified blocking lesions in ancient DNA on a single molecule level. Although we found clear evidence for blocking lesions in three out of four ancient samples, no more than 40% of the molecules were affected in any of the samples, indicating that such modifications are far less frequent in ancient DNA than previously thought.

Targeted investigation of the Neandertal genome by array-based sequence capture.

It is now possible to perform whole-genome shotgun sequencing as well as capture of specific genomic regions for extinct organisms. However, targeted resequencing of large parts of nuclear genomes has yet to be demonstrated for ancient DNA. Here we show that hybridization capture on microarrays can successfully recover more than a megabase of target regions from Neandertal DNA even in the presence of approximately 99.8% microbial DNA. Using this approach, we have sequenced approximately 14,000 protein-coding positions inferred to have changed on the human lineage since the last common ancestor shared with chimpanzees. By generating the sequence of one Neandertal and 50 present-day humans at these positions, we have identified 88 amino acid substitutions that have become fixed in humans since our divergence from the Neandertals.

A draft sequence of the Neandertal genome.

Neandertals, the closest evolutionary relatives of present-day humans, lived in large parts of Europe and western Asia before disappearing 30,000 years ago. We present a draft sequence of the Neandertal genome composed of more than 4 billion nucleotides from three individuals. Comparisons of the Neandertal genome to the genomes of five present-day humans from different parts of the world identify a number of genomic regions that may have been affected by positive selection in ancestral modern humans, including genes involved in metabolism and in cognitive and skeletal development. We show that Neandertals shared more genetic variants with present-day humans in Eurasia than with present-day humans in sub-Saharan Africa, suggesting that gene flow from Neandertals into the ancestors of non-Africans occurred before the divergence of Eurasian groups from each other.

A complete mtDNA genome of an early modern human from Kostenki, Russia.

The recovery of DNA sequences from early modern humans (EMHs) could shed light on their interactions with archaic groups such as Neandertals and their relationships to current human populations. However, such experiments are highly problematic because present-day human DNA frequently contaminates bones [1, 2]. For example, in a recent study of mitochondrial (mt) DNA from Neolithic European skeletons, sequence variants were only taken as authentic if they were absent or rare in the present population, whereas others had to be discounted as possible contamination [3, 4]. This limits analysis to EMH individuals carrying rare sequences and thus yields a biased view of the ancient gene pool. Other approaches of identifying contaminating DNA, such as genotyping all individuals who have come into contact with a sample, restrict analyses to specimens where this is possible [5, 6] and do not exclude all possible sources of contamination. By studying mtDNA in Neandertal remains, where contamination and endogenous DNA can be distinguished by sequence, we show that fragmentation patterns and nucleotide misincorporations can be used to gauge authenticity of ancient DNA sequences. We use these features to determine a complete mtDNA sequence from a approximately 30,000-year-old EMH from the Kostenki 14 site in Russia.

Removal of deaminated cytosines and detection of in vivo methylation in ancient DNA.

DNA sequences determined from ancient organisms have high error rates, primarily due to uracil bases created by cytosine deamination. We use synthetic oligonucleotides, as well as DNA extracted from mammoth and Neandertal remains, to show that treatment with uracil-DNA-glycosylase and endonuclease VIII removes uracil residues from ancient DNA and repairs most of the resulting abasic sites, leaving undamaged parts of the DNA fragments intact. Neandertal DNA sequences determined with this protocol have greatly increased accuracy. In addition, our results demonstrate that Neandertal DNA retains in vivo patterns of CpG methylation, potentially allowing future studies of gene inactivation and imprinting in ancient organisms.