Author Correction: A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

A single-cell and single-nucleus RNA-Seq toolbox for fresh and frozen human tumors.

Single-cell genomics is essential to chart tumor ecosystems. Although single-cell RNA-Seq (scRNA-Seq) profiles RNA from cells dissociated from fresh tumors, single-nucleus RNA-Seq (snRNA-Seq) is needed to profile frozen or hard-to-dissociate tumors. Each requires customization to different tissue and tumor types, posing a barrier to adoption. Here, we have developed a systematic toolbox for profiling fresh and frozen clinical tumor samples using scRNA-Seq and snRNA-Seq, respectively. We analyzed 216,490 cells and nuclei from 40 samples across 23 specimens spanning eight tumor types of varying tissue and sample characteristics. We evaluated protocols by cell and nucleus quality, recovery rate and cellular composition. scRNA-Seq and snRNA-Seq from matched samples recovered the same cell types, but at different proportions. Our work provides guidance for studies in a broad range of tumors, including criteria for testing and selecting methods from the toolbox for other tumors, thus paving the way for charting tumor atlases.

Integrated scRNA-Seq Identifies Human Postnatal Thymus Seeding Progenitors and Regulatory Dynamics of Differentiating Immature Thymocytes.

During postnatal life, thymopoiesis depends on the continuous colonization of the thymus by bone-marrow-derived hematopoietic progenitors that migrate through the bloodstream. The current understanding of the nature of thymic immigrants is largely based on data from pre-clinical models. Here, we employed single-cell RNA sequencing (scRNA-seq) to examine the immature postnatal thymocyte population in humans. Integration of bone marrow and peripheral blood precursor datasets identified two putative thymus seeding progenitors that varied in expression of CD7; CD10; and the homing receptors CCR7, CCR9, and ITGB7. Whereas both precursors supported T cell development, only one contributed to intrathymic dendritic cell (DC) differentiation, predominantly of plasmacytoid dendritic cells. Trajectory inference delineated the transcriptional dynamics underlying early human T lineage development, enabling prediction of transcription factor (TF) modules that drive stage-specific steps of human T cell development. This comprehensive dataset defines the expression signature of immature human thymocytes and provides a resource for the further study of human thymopoiesis.

Associations between host gene expression, the mucosal microbiome, and clinical outcome in the pelvic pouch of patients with inflammatory bowel disease.

BACKGROUND: Pouchitis is common after ileal pouch-anal anastomosis (IPAA) surgery for ulcerative colitis (UC). Similar to inflammatory bowel disease (IBD), both host genetics and the microbiota are implicated in its pathogenesis. We use the IPAA model of IBD to associate mucosal host gene expression with mucosal microbiomes and clinical outcomes. We analyze host transcriptomic data and 16S rRNA gene sequencing data from paired biopsies from IPAA patients with UC and familial adenomatous polyposis. To achieve power for a genome-wide microbiome-transcriptome association study, we use principal component analysis for transcript and clade reduction, and identify significant co-variation between clades and transcripts. RESULTS: Host transcripts co-vary primarily with biopsy location and inflammation, while microbes co-vary primarily with antibiotic use. Transcript-microbe associations are surprisingly modest, but the most strongly microbially-associated host transcript pattern is enriched for complement cascade genes and for the interleukin-12 pathway. Activation of these host processes is inversely correlated with Sutterella, Akkermansia, Bifidobacteria, and Roseburia abundance, and positively correlated with Escherichia abundance. CONCLUSIONS: This study quantifies the effects of inflammation, antibiotic use, and biopsy location upon the microbiome and host transcriptome during pouchitis. Understanding these effects is essential for basic biological insights as well as for well-designed and adequately-powered studies. Additionally, our study provides a method for profiling host-microbe interactions with appropriate statistical power using high-throughput sequencing, and suggests that cross-sectional changes in gut epithelial transcription are not a major component of the host-microbiome regulatory interface during pouchitis.

Pediatric Crohn disease patients exhibit specific ileal transcriptome and microbiome signature.

Interactions between the host and gut microbial community likely contribute to Crohn disease (CD) pathogenesis; however, direct evidence for these interactions at the onset of disease is lacking. Here, we characterized the global pattern of ileal gene expression and the ileal microbial community in 359 treatment-naive pediatric patients with CD, patients with ulcerative colitis (UC), and control individuals. We identified core gene expression profiles and microbial communities in the affected CD ilea that are preserved in the unaffected ilea of patients with colon-only CD but not present in those with UC or control individuals; therefore, this signature is specific to CD and independent of clinical inflammation. An abnormal increase of antimicrobial dual oxidase (DUOX2) expression was detected in association with an expansion of Proteobacteria in both UC and CD, while expression of lipoprotein APOA1 gene was downregulated and associated with CD-specific alterations in Firmicutes. The increased DUOX2 and decreased APOA1 gene expression signature favored oxidative stress and Th1 polarization and was maximally altered in patients with more severe mucosal injury. A regression model that included APOA1 gene expression and microbial abundance more accurately predicted month 6 steroid-free remission than a model using clinical factors alone. These CD-specific host and microbe profiles identify the ileum as the primary inductive site for all forms of CD and may direct prognostic and therapeutic approaches.

Correlation analysis of HOX, ErbB and IGFBP family gene expression in ovarian cancer.

Utilizing microarray gene expression data in cancer research possesses the ability to identify deregulated cellular pathways involved in malignant development. This study investigated the relationships of three gene families, HOX, ErbB and IGFBP, with regard to the development of ovarian cancer. These families were of interest because of similar chromosomal locations and their deregulated expression in ovarian cancer. Higher level statistics were used to differentially analyze microarray data in 65 ovarian samples to assess correlation and relationships among the gene families of interest. Fifteen genes in the three families were found to be significantly deregulated. Thirty-eight significant correlations were found within and between the genes of interest. Our data indicates that the significantly modeled relationships between HOX, ErbB and IGFBP gene pairs could provide insight into the underlying biological mechanisms in ovarian cancer.