Biology subjects

Sebra, R. P.

Publications and source records attributed to Sebra, R. P..

6 recordsLinked to original sources

bioRxiv2022

FLAIRR-seq: A novel method for single molecule resolution of near full-length immunoglobulin heavy chain repertoires

Current Adaptive Immune Receptor Repertoire Sequencing (AIRR-seq) strategies resolve expressed antibody (Ab) transcripts with limited resolution of the constant region. Here we present a novel near full-length AIRR-seq (FLAIRR-Seq) method that utilizes targeted amplification by 5 rapid amplification of cDNA ends (RACE), combined with single molecule, real-time sequencing to generate highly accurate (>Q40, 99.99%) IG heavy chain transcripts. FLAIRR-seq was benchmarked by comparing IG heavy chain variable (IGHV), diversity (IGHD), and joining (IGHJ) gene usage, complementarity-determining region 3 (CDR3) length, and somatic hypermutation to matched datasets generated with standard 5 RACE AIRR-seq and full-length isoform sequencing. Together these data demonstrate robust, unbiased FLAIRR-seq performance using RNA samples derived from peripheral blood mononuclear cells, purified B cells, and whole blood, which recapitulated results generated by commonly used methods, while additionally resolving novel IG heavy chain constant (IGHC) gene features. FLAIRR-seq data provides, for the first time, simultaneous, single-molecule characterization of IGHV, IGHD, IGHJ, and IGHC region genes and alleles, allele-resolved subisotype definition, and high-resolution identification of class-switch recombination within a clonal lineage. In conjunction with genomic sequencing and genotyping of IGHC genes, FLAIRR-seq of the IgM and IgG repertoires from 10 individuals resulted in the identification of 32 unique IGHC alleles, 28 (87%) of which were previously uncharacterized. Together, these data demonstrate the capabilities of FLAIRR-seq to characterize IGHV, IGHD, IGHJ, and IGHC gene diversity for the most comprehensive view of bulk expressed Ab repertoires to date.

immunology↗

bioRxiv2022

NKG2A and HLA-E define a novel alternative immune checkpoint axis in bladder cancer

PD-1/PD-L1-blockade immunotherapies have limited efficacy in the treatment of muscle-invasive bladder cancer (MIBC) and metastatic urothelial carcinoma. Here, we show that KLRC1 (NKG2A) expression associates with improved survival and responsiveness to PD-L1 blockade immunotherapy in CD8Ahigh bladder tumors. The loss of antigen presentation is a common mechanism for tumor escape in bladder cancer. NKG2A+ CD8 T cells are able to circumvent HLA-ABC loss through TCR-independent cytotoxicity, which is partly mediated by DNAM-1. In bladder tumors, NKG2A is acquired on a subset of PD-1+ CD8 T cells, alongside stronger tissue-residency memory features, TCR-independent cytotoxicity and evidence of recent proliferation. HLA-E is low but variably expressed on bladder tumors. When expressed, NKG2A+ CD8 T cell anti-tumor responses to HLA-ABC-deficient tumors are inhibited and partly restored upon NKG2A blockade. Overall, our study identifies an alternative path for CD8 T cell exhaustion, that is mediated by NKG2A upregulation and TCR-independent cytotoxicity.

immunology↗

bioRxiv2019

The impact of sequencing depth on the inferred taxonomic composition and AMR gene content of metagenomic samples

BackgroundShotgun metagenomics is increasingly used to characterise microbial communities, particularly for the investigation of antimicrobial resistance (AMR) in different animal and environmental contexts. There are many different approaches for inferring the taxonomic composition and AMR gene content of complex community samples from shotgun metagenomic data, but there has been little work establishing the optimum sequencing depth, data processing and analysis methods for these samples. In this study we used shotgun metagenomics and sequencing of cultured isolates from the same samples to address these issues. We sampled three potential environmental AMR gene reservoirs (pig caeca, river sediment, effluent) and sequenced samples with shotgun metagenomics at high depth ([~]200 million reads per sample). Alongside this, we cultured single-colony isolates of Enterobacteriaceae from the same samples and used hybrid sequencing (short- and long-reads) to create high-quality assemblies for comparison to the metagenomic data. To automate data processing, we developed an open-source software pipeline, ResPipe.\n\nResultsTaxonomic profiling was much more stable to sequencing depth than AMR gene content. 1 million reads per sample was sufficient to achieve <1% dissimilarity to the full taxonomic composition. However, at least 80 million reads per sample were required to recover the full richness of different AMR gene families present in the sample, and additional allelic diversity of AMR genes was still being discovered in effluent at 200 million reads per sample. Normalising the number of reads mapping to AMR genes using gene length and an exogenous spike of Thermus thermophilus DNA substantially changed the estimated gene abundance distributions. While the majority of genomic content from cultured isolates from effluent was recoverable using shotgun metagenomics, this was not the case for pig caeca or river sediment.\n\nConclusionsSequencing depth and profiling method can critically affect the profiling of polymicrobial animal and environmental samples with shotgun metagenomics. Both sequencing of cultured isolates and shotgun metagenomics can recover substantial diversity that is not identified using the other methods. Particular consideration is required when inferring AMR gene content or presence by mapping metagenomic reads to a database. ResPipe, the open-source software pipeline we have developed, is freely available (https://gitlab.com/hsgweon/ResPipe).

microbiology↗

bioRxiv2018

Reconstitution of eukaryotic chromosomes and manipulation of DNA N6-methyladenine alters chromatin and gene expression

DNA N6-adenine methylation (6mA) has recently been reported in diverse eukaryotes, spanning unicellular organisms to metazoans. Yet the functional significance of 6mA remains elusive due to its low abundance, difficulty of manipulation within native DNA, and lack of understanding of eukaryotic 6mA writers. Here, we report a novel DNA 6mA methyltransferase in ciliates, termed MTA1. The enzyme contains an MT-A70 domain but is phylogenetically distinct from all known RNA and DNA methyltransferases. Disruption of MTA1 in vivo leads to the genome-wide loss of 6mA in asexually growing cells and abolishment of the consensus ApT dimethylated motif. Genes exhibit subtle changes in chromatin organization or RNA expression upon loss of 6mA, depending on their starting methylation level. Mutants fail to complete the sexual cycle, which normally coincides with a peak of MTA1 expression. Thus, MTA1 functions in a developmental stage-specific manner. We determine the impact of 6mA on chromatin organization in vitro by reconstructing complete, full-length ciliate chromosomes harboring 6mA in native or ectopic positions. Using these synthetic chromosomes, we show that 6mA directly disfavors nucleosomes in vitro in a local, quantitative manner, independent of DNA sequence. Furthermore, the chromatin remodeler ACF can overcome this effect. Our study identifies a novel MT-A70 protein necessary for eukaryotic 6mA methylation and defines the impact of 6mA on chromatin organization using epigenetically defined synthetic chromosomes.\n\nHighlightsO_LIThe MT-A70 protein MTA1 mediates DNA N6-adenine methylation in Oxytricha\nC_LIO_LIMTA1 mutants exhibit subtle changes in nucleosome organization and transcription in vivo\nC_LIO_LI6mA directly disfavors nucleosome occupancy in natural and synthetic chromosomes in vitro\nC_LIO_LIDe novo synthesis of complete, epigenetically defined Oxytricha chromosomes\nC_LI

biochemistry↗

bioRxiv2017

Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

Biochemical studies of chromatin have typically used either artificial DNA templates with unnaturally high affinity for histones, or small genomic DNA fragments deprived of their cognate physical environment. It has thus been difficult to dissect chromatin structure and function within fully native DNA substrates. Here, we circumvent these limitations by exploiting the minimalist genome of the eukaryote Oxytricha trifallax, whose notably small ~3kb chromosomes mainly encode single genes. Guided by high-resolution epigenomic maps of nucleosome organization, transcription, and DNA N6-methyladenine (m6dA) locations, we reconstruct full-length Oxytricha chromosomes in vitro and use these synthetic facsimiles to dissect the influence of m6dA and histone post-translational modifications on nucleosome organization. We show that m6dA directly disfavors nucleosomes in a quantitative manner, leading to local decreases in nucleosome occupancy that are synergistic with histone acetylation. The effect of m6dA can be partially reversed by the action of an ATP-dependent chromatin remodeler. Furthermore, erasing m6dA marks from Oxytricha chromosomes leads to proportional increases in nucleosome occupancy across the genome. This work showcases Oxytricha chromosomes as powerful yet practical models for studying eukaryotic chromatin and transcription in the context of biologically relevant DNA substrates.\n\nHighlightsO_LIDe novo synthesis of complete, epigenetically defined Oxytricha chromosomes\nC_LIO_LIEpigenomic profiles of chromatin organization in Oxytrichas miniature chromosomes\nC_LIO_LIm6dA directly disfavors nucleosome occupancy in natural and synthetic chromosomes\nC_LIO_LIHistone acetylation and chromatin remodelers temper the impact of m6dA on chromatin\nC_LI

biochemistry↗

bioRxiv2017

Parallel evolution of two clades of a major Atlantic endemic Vibrio parahaemolyticus pathogen lineage by independent acquisition of related pathogenicity islands

Shellfish-transmitted Vibrio parahaemolyticus infections have recently increased from locations with historically low disease incidence, such as the Northeast United States (US). This change coincided with a bacterial population shift towards human pathogenic variants occurring in part through the introduction of several Pacific native lineages (ST36, ST43 and ST636) to near-shore areas off the Atlantic coast of the Northeast US. Concomitantly, ST631 emerged as a major endemic pathogen. Phylogenetic trees of clinical and environmental isolates indicated that two clades diverged from a common ST631 ancestor, and in each of these clades, a human pathogenic variant evolved independently through acquisition of distinct Vibrio pathogenicity islands (VPaI). These VPaI differ from each other and bear little resemblance to hemolysin-containing VPaI from isolates of the pandemic clonal complex. Clade I ST631 isolates either harbored no hemolysins, or contained a chromosome I-inserted island we call VPaI{beta} that encodes a type three secretion system (T3SS2{beta}) typical of Trh hemolysin-producers. The more clinically prevalent and clonal ST631 clade II had an island we call VPaI{gamma} that encodes both tdh and trh and that was inserted in chromosome II. VPaI{gamma} was derived from VPaI{beta} but with some additional acquired elements in common with VPaI carried by pandemic isolates, exemplifying the mosaic nature of pathogenicity islands. Genomics comparisons and amplicon assays identified VPaI{gamma}-type islands containing tdh inserted adjacent to the ure cluster in the three introduced Pacific and most other emergent lineages. that collectively cause 67% of Northeast US infections as of 2016.\n\nIMPORTANCEThe availability of three different hemolysin genotypes in the ST631 lineage provided a unique opportunity to employ genome comparisons to further our understanding of the processes underlying pathogen evolution. The fact that two different pathogenic clades arose in parallel from the same potentially benign lineage by independent VPaI acquisition is surprising considering the historically low prevalence of community members harboring VPaI in waters along the Northeast US Coast that could serve as the source of this material. This illustrates a possible predisposition of some lineages to not only acquire foreign DNA but also to become human pathogens. Whereas the underlying cause for the expansion of V. parahaemolyticus lineages harboring VPaI{gamma} along the US Atlantic coast and spread of this element to multiple lineages that underlies disease emergence is not known, this work underscores the need to define the environment factors that favor bacteria harboring VPaI in locations of emergent disease.

evolutionary biology↗