Life stage-specific poly(A) site selection regulated by Trypanosoma brucei DRBD18.

The kinetoplastid parasite, Trypanosoma brucei, undergoes a complex life cycle entailing slender and stumpy bloodstream forms in mammals and procyclic and metacyclic forms (MFs) in tsetse fly hosts. The numerous gene regulatory events that underlie T. brucei differentiation between hosts, as well as between active and quiescent stages within each host, take place in the near absence of transcriptional control. Rather, differentiation is controlled by RNA-binding proteins (RBPs) that associate with mRNA 3' untranslated regions (3'UTRs) to impact RNA stability and translational efficiency. DRBD18 is a multifunctional T. brucei RBP, shown to impact mRNA stability, translation, export, and processing. Here, we use single-cell RNAseq to characterize transcriptomic changes in cell populations that arise upon DRBD18 depletion, as well as to visualize transcriptome-wide alterations to 3'UTR length. We show that in procyclic insect stages, DRBD18 represses expression of stumpy bloodstream form and MF transcripts. Additionally, DRBD18 regulates the 3'UTR lengths of over 1,500 transcripts, typically promoting the use of distal polyadenylation sites, and thus the inclusion of 3'UTR regulatory elements. Remarkably, comparison of polyadenylation patterns in DRBD18 knockdowns with polyadenylation patterns in stumpy bloodstream forms shows numerous similarities, revealing a role for poly(A) site selection in developmental gene regulation, and indicating that DRBD18 controls this process for a set of transcripts. RNA immunoprecipitation supports a direct role for DRBD18 in poly(A) site selection. This report highlights the importance of alternative polyadenylation in T. brucei developmental control and identifies a critical RBP in this process.

Key transcription factors influence the epigenetic landscape to regulate retinal cell differentiation.

How the diverse neural cell types emerge from multipotent neural progenitor cells during central nervous system development remains poorly understood. Recent scRNA-seq studies have delineated the developmental trajectories of individual neural cell types in many neural systems including the neural retina. Further understanding of the formation of neural cell diversity requires knowledge about how the epigenetic landscape shifts along individual cell lineages and how key transcription factors regulate these changes. In this study, we dissect the changes in the epigenetic landscape during early retinal cell differentiation by scATAC-seq and identify globally the enhancers, enriched motifs, and potential interacting transcription factors underlying the cell state/type specific gene expression in individual lineages. Using CUT&Tag, we further identify the enhancers bound directly by four key transcription factors, Otx2, Atoh7, Pou4f2 and Isl1, including those dependent on Atoh7, and uncover the sequential and combinatorial interactions of these factors with the epigenetic landscape to control gene expression along individual retinal cell lineages such as retinal ganglion cells (RGCs). Our results reveal a general paradigm in which transcription factors collaborate and compete to regulate the emergence of distinct retinal cell types such as RGCs from multipotent retinal progenitor cells (RPCs).

Developmental constraint shaped genome evolution and erythrocyte loss in Antarctic fishes following paleoclimate change.

In the frigid, oxygen-rich Southern Ocean (SO), Antarctic icefishes (Channichthyidae; Notothenioidei) evolved the ability to survive without producing erythrocytes and hemoglobin, the oxygen-transport system of virtually all vertebrates. Here, we integrate paleoclimate records with an extensive phylogenomic dataset of notothenioid fishes to understand the evolution of trait loss associated with climate change. In contrast to buoyancy adaptations in this clade, we find relaxed selection on the genetic regions controlling erythropoiesis evolved only after sustained cooling in the SO. This pattern is seen not only within icefishes but also occurred independently in other high-latitude notothenioids. We show that one species of the red-blooded dragonfish clade evolved a spherocytic anemia that phenocopies human patients with this disease via orthologous mutations. The genomic imprint of SO climate change is biased toward erythrocyte-associated conserved noncoding elements (CNEs) rather than to coding regions, which are largely preserved through pleiotropy. The drift in CNEs is specifically enriched near genes that are preferentially expressed late in erythropoiesis. Furthermore, we find that the hematopoietic marrow of icefish species retained proerythroblasts, which indicates that early erythroid development remains intact. Our results provide a framework for understanding the interactions between development and the genome in shaping the response of species to climate change.

Comparative genomic analysis of a Shiga toxin-producing Escherichia coli (STEC) O145:H25 associated with a severe pediatric case of hemolytic uremic syndrome in Davidson County, Tennessee, US.

BACKGROUND: Shiga toxin-producing E. coli (STECs) are foodborne pathogens associated with bloody diarrhea and hemolytic uremic syndrome (HUS). Although the STEC O157 serogroup accounts for the highest number of infections, HUS-related complications and deaths, the STEC non-O157, as a group, accounts for a larger proportion of STEC infections and lower HUS cases. There is limited information available on how to recognize non-O157 serotypes associated with severe disease. The objectives of this study were to describe a patient with STEC non-O157 infection complicated with HUS and to conduct a comparative whole genome sequence (WGS) analysis among the patient's STEC clinical isolate and STEC O157 and non-O157 strains. RESULTS: The STEC O145:H25 strain EN1I-0044-2 was isolated from a pediatric patient with diarrhea, HUS and severe neurologic and cardiorespiratory complications, who was enrolled in a previously reported case-control study of acute gastroenteritis conducted in Davidson County, Tennessee in 2013. The strain EN1I-0044-2 genome sequence contained a chromosome and three plasmids. Two of the plasmids were similar to those present in O145:H25 strains whereas the third unique plasmid EN1I-0044-2_03 shared no similarity with other STEC plasmids, and it carried 23 genes of unknown function. Strain EN1I-0044-2, compared with O145:H25 and O157 serogroup strains shared chromosome- and plasmid-encoded virulence factors, including Shiga toxin, LEE type III secretion system, LEE effectors, SFP fimbriae, and additional toxins and colonization factors. CONCLUSIONS: A STEC O145:H25 strain EN1I-0044-2 was isolated from a pediatric patient with severe disease, including HUS, in Davidson County, TN. Phylogenetic and comparison WGS analysis provided evidence that strain EN1I-0044-2 closely resembles O145:H25, and confirmed an independent evolutionary path of STEC O145:H25 and O145:H28 serotypes. The strain EN1I-0044-2 virulence make up was similar to other O145:H25 and O157 serogroups. It carried stx2 and the LEE pathogenicity island, and additional colonization factors and enterotoxin genes. A unique feature of strain EN1I-0044-2 was the presence of plasmid pEN1I-0044-2_03 carrying genes with functions to be determined. Further studies will be necessary to elucidate the role that newly acquired genes by O145:H25 strains play in pathogenesis, and to determine if they may serve as genetic markers of severe disease.

C11orf95-RELA fusions drive oncogenic NF-κB signalling in ependymoma.

Members of the nuclear factor-κB (NF-κB) family of transcriptional regulators are central mediators of the cellular inflammatory response. Although constitutive NF-κB signalling is present in most human tumours, mutations in pathway members are rare, complicating efforts to understand and block aberrant NF-κB activity in cancer. Here we show that more than two-thirds of supratentorial ependymomas contain oncogenic fusions between RELA, the principal effector of canonical NF-κB signalling, and an uncharacterized gene, C11orf95. In each case, C11orf95-RELA fusions resulted from chromothripsis involving chromosome 11q13.1. C11orf95-RELA fusion proteins translocated spontaneously to the nucleus to activate NF-κB target genes, and rapidly transformed neural stem cells--the cell of origin of ependymoma--to form these tumours in mice. Our data identify a highly recurrent genetic alteration of RELA in human cancer, and the C11orf95-RELA fusion protein as a potential therapeutic target in supratentorial ependymoma.

Global Genome Conformational Programming during Neuronal Development Is Associated with CTCF and Nuclear FGFR1-The Genome Archipelago Model.

During the development of mouse embryonic stem cells (ESC) to neuronal committed cells (NCC), coordinated changes in the expression of 2851 genes take place, mediated by the nuclear form of FGFR1. In this paper, widespread differences are demonstrated in the ESC and NCC inter- and intra-chromosomal interactions, chromatin looping, the formation of CTCF- and nFGFR1-linked Topologically Associating Domains (TADs) on a genome-wide scale and in exemplary HoxA-D loci. The analysis centered on HoxA cluster shows that blocking FGFR1 disrupts the loop formation. FGFR1 binding and genome locales are predictive of the genome interactions; likewise, chromatin interactions along with nFGFR1 binding are predictive of the genome function and correlate with genome regulatory attributes and gene expression. This study advances a topologically integrated genome archipelago model that undergoes structural transformations through the formation of nFGFR1-associated TADs. The makeover of the TAD islands serves to recruit distinct ontogenic programs during the development of the ESC to NCC.

Correction to: H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

The original article can be found online.

H3.3 K27M depletion increases differentiation and extends latency of diffuse intrinsic pontine glioma growth in vivo.

Histone H3 K27M mutation is the defining molecular feature of the devastating pediatric brain tumor, diffuse intrinsic pontine glioma (DIPG). The prevalence of histone H3 K27M mutations indicates a critical role in DIPGs, but the contribution of the mutation to disease pathogenesis remains unclear. We show that knockdown of this mutation in DIPG xenografts restores K27M-dependent loss of H3K27me3 and delays tumor growth. Comparisons of matched DIPG xenografts with and without K27M knockdown allowed identification of mutation-specific effects on the transcriptome and epigenome. The resulting transcriptional changes recapitulate expression signatures from K27M primary DIPG tumors and are strongly enriched for genes associated with nervous system development. Integrated analysis of ChIP-seq and expression data showed that genes upregulated by the mutation are overrepresented in apparently bivalent promoters. Many of these targets are associated with more immature differentiation states. Expression profiles indicate K27M knockdown decreases proliferation and increases differentiation within lineages represented in DIPG. These data suggest that K27M-mediated loss of H3K27me3 directly regulates a subset of genes by releasing poised promoters, and contributes to tumor phenotype and growth by limiting differentiation. The delayed tumor growth associated with knockdown of H3 K27M provides evidence that this highly recurrent mutation is a relevant therapeutic target.

Germline Mutations in Predisposition Genes in Pediatric Cancer.

BACKGROUND: The prevalence and spectrum of predisposing mutations among children and adolescents with cancer are largely unknown. Knowledge of such mutations may improve the understanding of tumorigenesis, direct patient care, and enable genetic counseling of patients and families. METHODS: In 1120 patients younger than 20 years of age, we sequenced the whole genomes (in 595 patients), whole exomes (in 456), or both (in 69). We analyzed the DNA sequences of 565 genes, including 60 that have been associated with autosomal dominant cancer-predisposition syndromes, for the presence of germline mutations. The pathogenicity of the mutations was determined by a panel of medical experts with the use of cancer-specific and locus-specific genetic databases, the medical literature, computational predictions, and second hits identified in the tumor genome. The same approach was used to analyze data from 966 persons who did not have known cancer in the 1000 Genomes Project, and a similar approach was used to analyze data from an autism study (from 515 persons with autism and 208 persons without autism). RESULTS: Mutations that were deemed to be pathogenic or probably pathogenic were identified in 95 patients with cancer (8.5%), as compared with 1.1% of the persons in the 1000 Genomes Project and 0.6% of the participants in the autism study. The most commonly mutated genes in the affected patients were TP53 (in 50 patients), APC (in 6), BRCA2 (in 6), NF1 (in 4), PMS2 (in 4), RB1 (in 3), and RUNX1 (in 3). A total of 18 additional patients had protein-truncating mutations in tumor-suppressor genes. Of the 58 patients with a predisposing mutation and available information on family history, 23 (40%) had a family history of cancer. CONCLUSIONS: Germline mutations in cancer-predisposing genes were identified in 8.5% of the children and adolescents with cancer. Family history did not predict the presence of an underlying predisposition syndrome in most patients. (Funded by the American Lebanese Syrian Associated Charities and the National Cancer Institute.).

Genome-wide segregation of single nucleotide and structural variants into single cancer cells.

BACKGROUND: Single-cell genome sequencing provides high-resolution details of the clonal genomic modifications that occur during cancer initiation, progression, and ongoing evolution as patients undergo treatment. One limitation of current single-cell sequencing strategies is a suboptimal capacity to detect all classes of single-nucleotide and structural variants in the same cells. RESULTS: Here we present a new approach for determining comprehensive variant profiles of single cells using a microfluidic amplicon-based strategy to detect structural variant breakpoint sequences instead of using relative read depth to infer copy number changes. This method can reconstruct the clonal architecture and mutational history of a malignancy using all classes and sizes of somatic variants, providing more complete details of the temporal changes in mutational classes and processes that led to the development of a malignant neoplasm. Using this approach, we interrogated cells from a patient with leukemia, determining that processes producing structural variation preceded single nucleotide changes in the development of that malignancy. CONCLUSIONS: All classes and sizes of genomic variants can be efficiently detected in single cancer cells using our new method, enabling the ordering of distinct classes of mutations during tumor evolution.

Targetable kinase-activating lesions in Ph-like acute lymphoblastic leukemia.

BACKGROUND: Philadelphia chromosome-like acute lymphoblastic leukemia (Ph-like ALL) is characterized by a gene-expression profile similar to that of BCR-ABL1-positive ALL, alterations of lymphoid transcription factor genes, and a poor outcome. The frequency and spectrum of genetic alterations in Ph-like ALL and its responsiveness to tyrosine kinase inhibition are undefined, especially in adolescents and adults. METHODS: We performed genomic profiling of 1725 patients with precursor B-cell ALL and detailed genomic analysis of 154 patients with Ph-like ALL. We examined the functional effects of fusion proteins and the efficacy of tyrosine kinase inhibitors in mouse pre-B cells and xenografts of human Ph-like ALL. RESULTS: Ph-like ALL increased in frequency from 10% among children with standard-risk ALL to 27% among young adults with ALL and was associated with a poor outcome. Kinase-activating alterations were identified in 91% of patients with Ph-like ALL; rearrangements involving ABL1, ABL2, CRLF2, CSF1R, EPOR, JAK2, NTRK3, PDGFRB, PTK2B, TSLP, or TYK2 and sequence mutations involving FLT3, IL7R, or SH2B3 were most common. Expression of ABL1, ABL2, CSF1R, JAK2, and PDGFRB fusions resulted in cytokine-independent proliferation and activation of phosphorylated STAT5. Cell lines and human leukemic cells expressing ABL1, ABL2, CSF1R, and PDGFRB fusions were sensitive in vitro to dasatinib, EPOR and JAK2 rearrangements were sensitive to ruxolitinib, and the ETV6-NTRK3 fusion was sensitive to crizotinib. CONCLUSIONS: Ph-like ALL was found to be characterized by a range of genomic alterations that activate a limited number of signaling pathways, all of which may be amenable to inhibition with approved tyrosine kinase inhibitors. Trials identifying Ph-like ALL are needed to assess whether adding tyrosine kinase inhibitors to current therapy will improve the survival of patients with this type of leukemia. (Funded by the American Lebanese Syrian Associated Charities and others.).

Robust Performance of SARS-CoV-2 Whole-Genome Sequencing from Wastewater with a Nonselective Virus Concentration Method.

The sequencing of human virus genomes from wastewater samples is an efficient method for tracking viral transmission and evolution at the community level. However, this requires the recovery of viral nucleic acids of high quality. We developed a reusable tangential-flow filtration system to concentrate and purify viruses from wastewater for genome sequencing. A pilot study was conducted with 94 wastewater samples from four local sewersheds, from which viral nucleic acids were extracted, and the whole genome of severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) was sequenced using the ARTIC V4.0 primers. Our method yielded a high probability (0.9) of recovering complete or near-complete SARS-CoV-2 genomes (>90% coverage at 10× depth) from wastewater when the COVID-19 incidence rate exceeded 33 cases per 100 000 people. The relative abundances of sequenced SARS-CoV-2 variants followed the trends observed from patient-derived samples. We also identified SARS-CoV-2 lineages in wastewater that were underrepresented or not present in the clinical whole-genome sequencing data. The developed tangential-flow filtration system can be easily adopted for the sequencing of other viruses in wastewater, particularly those at low concentrations.

Core needle biopsy wash as a tool for acquiring additional diagnostic material for laboratory testing.

AIMS: To determine if a simple prewash step added to the processing workflow of tissue procurement by a core needle biopsy device will recover enough cells to expand the laboratory testing armamentarium. METHODS: Tissue was obtained from unfixed resection specimens using a core needle device and washed in a buffered solution before fixation. This creates a liquid aliquot from which dislodged cells can be kept and separated from the tissue specimen, the latter of which can then undergo traditional formalin-fixed, paraffin-embedded processing. RESULTS: Cells dislodged from the tissue during the biopsy procedure are recoverable, are representative of the tissue section and of sufficient quantities for additional laboratory testing. CONCLUSIONS: The core needle biopsy wash is an under-recognised and underutilised approach to extending the diagnostic capabilities of the limited amount of targeted material obtained during this common procedure. The ability to recover supplemental amounts of diagnostic material yields great potential as a substrate for a multitude of current and developing laboratory assays.

Transposon transgenesis in Xenopus.

Transposon-mediated integration strategies in Xenopus offer simple and robust methods for the generation of germline transgenic animals. Co-injection of fertilized one-cell embryos with plasmid DNA harboring a transposon transgene and synthetic mRNA encoding the cognate transposase enzyme results in mosaic integration of the transposon at early cleavage stages that are frequently passed through the germline in the adult animal. Micro-injection of fertilized embryos is a routine procedure used by many laboratories that use Xenopus as a developmental model and, as such, the transposon transgenesis method can be performed without additional equipment or specialized methodologies. The methods for injecting Xenopus embryos are well documented in the literature so here we provide a step-by-step guide to other aspects of transposon transgenesis, including screening mosaic founders for germline transmission of the transgene and general husbandry considerations related to management of populations of transgenic frogs.

Generation of mouse-human chimeric embryos.

Naive human pluripotent stem cells (hPSCs) can be used to generate mature human cells of all three germ layers in mouse-human chimeric embryos. Here, we describe a protocol for generating mouse-human chimeric embryos by injecting naive hPSCs converted from the primed state. Primed hPSCs are treated with a mammalian target of rapamycin inhibitor (Torin1) for 3 h and dissociated to single cells, which are plated on mouse embryonic fibroblasts in 2iLI medium, a condition essentially the same for culturing mouse embryonic stem cells. After 3-4 d, bright, dome-shaped colonies with mouse embryonic stem cell morphology are passaged in 2iLI medium. Established naive hPSCs are injected into mouse blastocysts, which produce E17.5 mouse embryos containing 0.1-4.0% human cells as quantified by next-generation sequencing of 18S ribosomal DNA amplicons. The protocol is suitable for studying the development of hPSCs in mouse embryos and may facilitate the generation of human cells, tissues and organs in animals.

Single cell transcriptomics reveals lineage trajectory of retinal ganglion cells in wild-type and Atoh7-null retinas.

Atoh7 has been believed to be essential for establishing the retinal ganglion cell (RGC) lineage, and Pou4f2 and Isl1 are known to regulate RGC specification and differentiation. Here we report our further study of the roles of these transcription factors. Using bulk RNA-seq, we identify genes regulated by the three transcription factors, which expand our understanding of the scope of downstream events. Using scRNA-seq on wild-type and mutant retinal cells, we reveal a transitional cell state of retinal progenitor cells (RPCs) co-marked by Atoh7 and other genes for different lineages and shared by all early retinal lineages. We further discover the unexpected emergence of the RGC lineage in the absence of Atoh7. We conclude that competence of RPCs for different retinal fates is defined by lineage-specific genes co-expressed in the transitional state and that Atoh7 defines the RGC competence and collaborates with other factors to shepherd transitional RPCs to the RGC lineage.

Integrative Genomic Analysis of Pediatric Myeloid-Related Acute Leukemias Identifies Novel Subtypes and Prognostic Indicators.

Genomic characterization of pediatric patients with acute myeloid leukemia (AML) has led to the discovery of somatic mutations with prognostic implications. Although gene-expression profiling can differentiate subsets of pediatric AML, its clinical utility in risk stratification remains limited. Here, we evaluate gene expression, pathogenic somatic mutations, and outcome in a cohort of 435 pediatric patients with a spectrum of pediatric myeloid-related acute leukemias for biological subtype discovery. This analysis revealed 63 patients with varying immunophenotypes that span a T-lineage and myeloid continuum designated as acute myeloid/T-lymphoblastic leukemia (AMTL). Within AMTL, two patient subgroups distinguished by FLT3-ITD and PRC2 mutations have different outcomes, demonstrating the impact of mutational composition on survival. Across the cohort, variability in outcomes of patients within isomutational subsets is influenced by transcriptional identity and the presence of a stem cell-like gene-expression signature. Integration of gene expression and somatic mutations leads to improved risk stratification. SIGNIFICANCE: Immunophenotype and somatic mutations play a significant role in treatment approach and risk stratification of acute leukemia. We conducted an integrated genomic analysis of pediatric myeloid malignancies and found that a combination of genetic and transcriptional readouts was superior to immunophenotype and genomic mutations in identifying biological subtypes and predicting outcomes. This article is highlighted in the In This Issue feature, p. 549.

A polygenic score for acute vaso-occlusive pain in pediatric sickle cell disease.

Individuals with monogenic disorders can experience variable phenotypes that are influenced by genetic variation. To investigate this in sickle cell disease (SCD), we performed whole-genome sequencing (WGS) of 722 individuals with hemoglobin HbSS or HbSß0-thalassemia from Baylor College of Medicine and from the St. Jude Children's Research Hospital Sickle Cell Clinical Research and Intervention Program (SCCRIP) longitudinal cohort study. We developed pipelines to identify genetic variants that modulate sickle hemoglobin polymerization in red blood cells and combined these with pain-associated variants to build a polygenic score (PGS) for acute vaso-occlusive pain (VOP). Overall, we interrogated the α-thalassemia deletion -α3.7 and 133 candidate single-nucleotide polymorphisms (SNPs) across 66 genes for associations with VOP in 327 SCCRIP participants followed longitudinally over 6 years. Twenty-one SNPs in 9 loci were associated with VOP, including 3 (BCL11A, MYB, and the ß-like globin gene cluster) that regulate erythrocyte fetal hemoglobin (HbF) levels and 6 (COMT, TBC1D1, KCNJ6, FAAH, NR3C1, and IL1A) that were associated previously with various pain syndromes. An unweighted PGS integrating all 21 SNPs was associated with the VOP event rate (estimate, 0.35; standard error, 0.04; P = 5.9 × 10-14) and VOP event occurrence (estimate, 0.42; standard error, 0.06; P = 4.1 × 10-13). These associations were stronger than those of any single locus. Our findings provide insights into the genetic modulation of VOP in children with SCD. More generally, we demonstrate the utility of WGS for investigating genetic contributions to the variable expression of SCD-associated morbidities.

Remobilization of Tol2 transposons in Xenopus tropicalis.

BACKGROUND: The Class II DNA transposons are mobile genetic elements that move DNA sequence from one position in the genome to another. We have previously demonstrated that the naturally occurring Tol2 element from Oryzias latipes efficiently integrates its corresponding non-autonomous transposable element into the genome of the diploid frog, Xenopus tropicalis. Tol2 transposons are stable in the frog genome and are transmitted to the offspring at the expected Mendelian frequency. RESULTS: To test whether Tol2 transposons integrated in the Xenopus tropicalis genome are substrates for remobilization, we injected in vitro transcribed Tol2 mRNA into one-cell embryos harbouring a single copy of a Tol2 transposon. Integration site analysis of injected embryos from two founder lines showed at least one somatic remobilization event per embryo. We also demonstrate that the remobilized transposons are transmitted through the germline and re-integration can result in the generation of novel GFP expression patterns in the developing tadpole. Although the parental line contained a single Tol2 transposon, the resulting remobilized tadpoles frequently inherit multiple copies of the transposon. This is likely to be due to the Tol2 transposase acting in discrete blastomeres of the developing injected embryo during the cell cycle after DNA synthesis but prior to mitosis. CONCLUSIONS: In this study, we demonstrate that single copy Tol2 transposons integrated into the Xenopus tropicalis genome are effective substrates for excision and random re-integration and that the remobilized transposons are transmitted through the germline. This is an important step in the development of 'transposon hopping' strategies for insertional mutagenesis, gene trap and enhancer trap screens in this highly tractable developmental model organism.

Pathogenic genetic variations of C. acnes are associated with clinically relevant orthopedic shoulder infections.

Many surgeons continue to face the clinical dilemma of interpreting a positive aspiration or unexpected positive Cutibacterium acnes (C. acnes) culture. There are factors that complicate the interpretation of positive cultures including variations in both frequency of false positive cultures and virulence properties. As indices of virulence, hemolytic strains, from previously confirmed clinically infected shoulders, were compared with non-hemolytic isolates determined to be contaminants, by RNA-sequencing (RNA-Seq). Six C. acnes isolates from patients who underwent revision total shoulder arthroplasty (TSA) were identified based on previously described infection criteria. Three C. acnes isolates from each group underwent RNA-Seq. Differential gene expression analysis, principal component analysis (PCA), and heatmap analysis were used to determine the gene variation and patterning between the definite infection and probable contaminant isolates. Differential gene expression analysis identified genes that were differentially expressed between the isolates classified as definite infection and isolates classified as probable contaminants. PCA using a 500 gene subset of identified genes was able to find combinations of these genes that separated out the definite infection and probable contaminants isolates. The heatmap demonstrated similar gene expression in the three Definite Infections isolates, and significantly different expression when compared with the probable contaminant isolates. Clinical significance: C. acnes revision TSA isolates classified as definite infection and probable contaminant demonstrated a similar gene expression pattern to each respective group and different gene expression pattern when compared between groups. These findings indicate distinct differences in C. acnes strains associated with clinically relevant orthopedic TSA infections.