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The functional annotation of gene lists is a common analysis routine required for most genomics experiments, and bioinformatics core facilities must support these analyses. In contrast to methods such as the quantitation of RNA-Seq reads or differential expression analysis, our research group noted a lack of consensus in our preferred approaches to functional annotation. To investigate this observation, we selected 4 experiments that represent a range of experimental designs encountered by our cores and analyzed those data with 6 tools used by members of the Association of Biomolecular Resource Facilities (ABRF) Genomic Bioinformatics Research Group (GBIRG). To facilitate comparisons between tools, we focused on a single biological result for each experiment. These results were represented by a gene set, and we analyzed these gene sets with each tool considered in our study to map the result to the annotation categories presented by each tool. In most cases, each tool produces data that would facilitate identification of the selected biological result for each experiment. For the exceptions, Fisher’s exact test parameters could be adjusted to detect the result. Because Fisher’s exact test is used by many functional annotation tools, we investigated input parameters and demonstrate that, while background set size is unlikely to have a significant impact on the results, the numbers of differentially expressed genes in an annotation category and the total number of differentially expressed genes under consideration are both critical parameters that may need to be modified during analyses. In addition, we note that differences in the annotation categories tested by each tool, as well as the composition of those categories, can have a significant impact on results.

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment. Woermann and colleagues describe a deaminase-independent function for APOBEC3A in initiating chromosomal instability and STING-dependent metastasis in human and mouse pancreatic cancer.

Chromosomal instability (CIN), a hallmark of cancer, has been associated with a highly metastatic phenotype in numerous cancers. Mechanisms by which these genomic events occur during the lifespan of a neoplasm have yet to be fully delineated. In our study we identified a novel function for APOBEC3A (A3A), a primate-specific cytidine deaminase upregulated in multiple cancers including Pancreatic ductal adenocarcinoma (PDAC), in the initiation of CIN.We demonstrate that A3A is not routinely detected in non-diseased pancreata; however, expression was detected in the epithelial and stromal compartments from patients with chronic pancreatitis and in precursor lesions of PDAC and is further significantly increased in invasive neoplasia. Strikingly, we found that A3A expression but not A3A-induced mutagenesis, was associated with a significantly reduced overall survival in PDAC. By employing a variety of genetic tools, we identified a novel function for A3A in initiating CIN, underscoring its potential role in driving the observed early metastatic propensity in PDAC. Using a series of in vivo and in vitro models, we demonstrate that A3A-induced CIN leads to aggressive cancer, featuring enhanced, STING-dependent, distant organ seeding and metastatic growth. As a consequence of aberrant A3A function and CIN-mediated upregulation of STING, NfkB and Stat3 pathways were activated. More importantly, all these effects were independent of the deaminase domain, underscoring a novel role of A3A beyond its established canonical function. Remarkably, in a novel autochthonous murine model of PDA expressing human A3A, we identified numerous copy number changes, homologous to those altered in A3A overexpressing patient tumors, including deletions in DNA repair pathway genes. While we showed that A3A-expressing PDAC cells do not fully phenocopy a BRCA mutation-like phenotype, A3A-expressing PDAC cells are exceptionally sensitive to PARP inhibition and DNA cross linking agents, recapitulating a state of “chemical BRCAness”. The combined effect of deletions in these genes has yet to be ascertained, and we hypothesize that these losses further add to ongoing DNA damage overall in PDAC. In summary, our study shows, that A3A drives metastasis and specific deletions through a deaminase-independent initiation of CIN, with potential implications for targeted treatment strategies in pancreatic cancer. Citation Format: Sonja Maria Woermann, Amy Zhang, Fredrik I. Thege, Chris Gates, Reuben S. Harris, Susan R. Ross, Faiyaz Notta, Anirban Maitra, Andrew D. Rhim. APOBEC3A a causative agent in cancer-related chromosomal instability and STING driven metastasis [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1506.

Adenosine-to-Inosine (A-to-I) RNA editing is mediated by Adenosine deaminase acting on RNA (Adar) enzymes. The resulting A-to-I edit in RNA alters its post-transcriptional processing making Adar1 a critical regulator of gene expression. In line with its role in gene regulation, Adar1 has been implicated in development and organogenesis. We investigated the role of Adar1 in pancreatic development and homeostasis, and observed that though Adar1 is not required for pancreatic development, it is essential for pancreatic homeostasis. We observed that loss of Adar1 can stimulate a Mavs-mediated innate immune pathway and results in influx of immune cells in the pancreas. Pancreatic tumors are notorious for their immunosuppressive microenvironment. Since loss of Adar1 can activate a Mavs-mediated innate immune pathway, we next investigated the role of ADAR1 in human pancreatic ductal adenocarcinoma cell lines. We developed a bioinformatics pipeline, Cancer RSNV (RNA Single Nucleotide Variant) Identifier and Annotator (CRIDA) to identify A-to-I RNA edits in pancreatic cancer and observed wide-spread ADAR1-mediated RNA editing in pancreatic cancer cells. We analyzed genes that are differentially regulated between ADAR1 knockdown and control cells, and observed a differential immune signaling. Together, our data suggests that Adar1 is important for pancreatic homeostasis and tumor biology. Citation Format: Dhwani Rupani, Robert Cowan, Chris Gates, Florencia McAllister, Andrew Rhim. Adar1 impacts pancreatic homeostasis and pancreatic tumor biology [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 1332.

Despite efforts in understanding its underlying mechanisms, the etiology of chromosomal instability (CIN) remains unclear for many tumor types. Here, we identify CIN initiation as a previously undescribed function for APOBEC3A (A3A), a cytidine deaminase upregulated across cancer types. Using genetic mouse models of pancreatic ductal adenocarcinoma (PDA) and genomics analyses in human tumor cells we show that A3A-induced CIN leads to aggressive tumors characterized by enhanced early dissemination and metastasis in a STING-dependent manner and independently of the canonical deaminase functions of A3A. We show that A3A upregulation recapitulates numerous copy number alterations commonly observed in patients with PDA, including co-deletions in DNA repair pathway genes, which in turn render these tumors susceptible to poly (ADP-ribose) polymerase inhibition. Overall, our results demonstrate that A3A plays an unexpected role in PDA as a specific driver of CIN, with significant effects on disease progression and treatment.

Over the last decade, the cost of -omics data creation has decreased 10-fold, whereas the need for analytical support for those data has increased exponentially. Consequently, bioinformaticians face a second wave of challenges: novel applications of existing approaches (e.g., single-cell RNA sequencing), integration of -omics data sets of differing size and scale (e.g., spatial transcriptomics), as well as novel computational and statistical methods, all of which require more sophisticated pipelines and data management. Nonetheless, bioinformatics cores are often asked to operate under primarily a cost-recovery model, with limited institutional support. Seeing the need to assess bioinformatics core operations, the Association of Biomolecular Resource Facilities Genomics Bioinformatics Research Group conducted a survey to answer questions about staffing, services, financial models, and challenges to better understand the challenges bioinformatics core facilities are currently faced with and will need to address going forward. Of the respondent groups, we chose to focus on the survey data from smaller cores, which made up the majority. Although all cores indicated similar challenges in terms of changing technologies and analysis needs, small cores tended to have the added challenge of funding their operations largely through cost-recovery models with heavy administrative burdens.

This chapter contains a step-by-step protocol for identifying somatic SNPs and small Indels from next-generation sequencing data of tumor samples and matching normal samples. The workflow presented here is largely based on the Broad Institute's "Best Practices" guidelines and makes use of their Genome Analysis Toolkit (GATK) platform. Variants are annotated with population allele frequencies and curated resources such as GnomAD and ClinVar and curated effect predictions from dbNSFP using VCFtools, SnpEff, and SnpSift.

The performance of high-contrast AO instruments (GPI, SPHERE, ScEXAO, MagAO) and other systems that operate at visible wavelengths can be severely hampered by control system latencies and temporal wavefront errors. In high-contrast systems, temporal errors and delays are manifest as high spatial frequency wavefront residuals that scatter light into the controllable region of the PSF and diminish contrast, an effect that is particularly severe when atmospheric coherence times are short. Solutions that have been proposed include lower latency electronics, deformable mirrors with lower mechanical response times, and specialized control algorithms such as predictive control. These advancements will be necessary for achieving the latency goals of high actuator count systems on future Extremely Large Telescopes (ELTs), including NFIRAOS+ and PFI on the Thirty Meter Telescope, upgrading the performance of existing highcontrast systems, and pushing adaptive optics to visible wavelengths. LLAMAS (Low-Latency Adaptive Optical Mirror System) is a fully funded adaptive optics system at the Lawrence Livermore National Laboratory site that will test these techniques in an integrated, real time, closed-loop AO system. With a total system latency goal of ~100 microseconds (including mechanical response time, not including frame integration), LLAMAS will achieve an order of magnitude improvement in AO system latencies over the current generation of high-contrast AO systems. The woofer/tweeter architecture will incorporate a 492-actuator Boston Micromachines MEMS device mapping 24 actuators across a circular pupil. The tweeter mirror will be paired with a specialized low-latency driver, delivering less than 40 microseconds electronic and mechanical latency (10 – 90%). The real-time control computer will utilize the computationally efficient Fourier Transform Reconstructor with a predictive Kalman filter with a goal of completing all computations and reconstructing the wavefront in less than 20 microseconds. LLAMAS will be fully integrated with a 21×21 lenslet Shack-Hartmann sensor by January 2019. These proceedings describe the LLAMAS design, characterize the performance of its low-latency componentry, and discuss the relevance of the design for future high-contrast, visiblelight, and high actuator count AO systems on ELTs.

Colorectal cancer arises in part from the cumulative effects of multiple gene lesions. Recent studies in selected cancer types have revealed significant intra-tumor genetic heterogeneity and highlighted its potential role in disease progression and resistance to therapy. We hypothesized the existence of significant intra-tumor genetic heterogeneity in rectal cancers involving variations in localized somatic mutations and copy number abnormalities. Two or three spatially disparate regions from each of six rectal tumors were dissected and subjected to next-generation whole exome DNA sequencing, Oncoscan SNP arrays, and targeted confirmatory sequencing and analysis. The resulting data were integrated to define subclones using SciClone. Mutant-allele tumor heterogeneity (MATH) scores, mutant allele frequency correlation, and mutation percent concordance were calculated, and copy number analysis including measurement of correlation between samples was performed. Somatic mutations profiles in individual cancers were similar to prior studies, with some variants found in previously reported significantly mutated genes and many patient-specific mutations in each tumor. Significant intra-tumor heterogeneity was identified in the spatially disparate regions of individual cancers. All tumors had some heterogeneity but the degree of heterogeneity was quite variable in the samples studied. We found that 67–97% of exonic somatic mutations were shared among all regions of an individual’s tumor. The SciClone computational method identified 2 to 8 shared and unshared subclones in the spatially disparate areas in each tumor. MATH scores ranged from 7 to 41. Allele frequency correlation scores ranged from R2 = 0.69 to 0.96. Measurements of correlation between samples for copy number changes varied from R2 = 0.74 to 0.93. All tumors had some heterogeneity, but the degree was highly variable in the samples studied. The occurrence of significant intra-tumor heterogeneity may allow selected tumors to have a genetic reservoir to draw from in their evolutionary response to therapy and other challenges.

The spine is a common site of metastatic spread of many cancers, causing debilitating pain and suffering. Treatment of spinal metastases is limited by resistance to radiation, chemotherapy, and proximity to the spinal cord. It is currently not known what causes the high incidence of spinal metastases, yet theories have been proposed, like the venous metastatic spread theory (Batson, 1940) and the “seed and soil” hypothesis (Paget, 1889), postulating that factors intrinsic to the tumor cells or to the microenvironment determine the location of cancer dissemination. Recent advances in high throughput sequencing allow for in depth analyses of the molecular signatures of tumors. To identify if there are intrinsic genetic alterations common to cancer cells that disseminate into the spine, we sequenced the exome of metastatic tumor cells harvested from the vertebrae of 9 patients with different primary tumors: carcinomas and sarcomas. Exome sequencing was performed using the HiSeq 4000 (Illumina) platform on DNA from tumor cell lines and control blood or bone marrow. Data was analyzed at the University of Michigan Bioinformatics Core and variants were called using the VarScan method (http://varscan.sourceforge.net/) in somatic mode. This analysis identified a total of 2366 genes with high impact mutations (888–1026 per sample); of these, 232 genes are common to all patients analyzed. Ninety-six of the identified genes (4%) are included in the Catalogue of Somatic Mutations in Cancer, and of these, seven: ACSL6, ACVR1, ALK, FGFR2, HSP90AA1, PTPN6 and PTPRB, have high impact mutations in all 9 patients with spinal metastatic disease. Pathway analysis of genes mutated in 5 or more patients shows significant overrepresentation of 41 KEGG pathways including TGFb, HIF1-a, VEGF, Wnt and Estrogen signaling pathways. Ongoing experiments are performed to validate the sequencing analysis and characterize functional consequences of the common mutations identified.

We report progress assembling the parts list for chromosome 17 and illustrate the various processes that we have developed to integrate available data from diverse genomic and proteomic knowledge bases. As primary resources, we have used GPMDB, neXtProt, PeptideAtlas, Human Protein Atlas (HPA), and GeneCards. All sites share the common resource of Ensembl for the genome modeling information. We have defined the chromosome 17 parts list with the following information: 1169 protein-coding genes, the numbers of proteins confidently identified by various experimental approaches as documented in GPMDB, neXtProt, PeptideAtlas, and HPA, examples of typical data sets obtained by RNASeq and proteomic studies of epithelial derived tumor cell lines (disease proteome) and a normal proteome (peripheral mononuclear cells), reported evidence of post-translational modifications, and examples of alternative splice variants (ASVs). We have constructed a list of the 59 "missing" proteins as well as 201 proteins that have inconclusive mass spectrometric (MS) identifications. In this report we have defined a process to establish a baseline for the incorporation of new evidence on protein identification and characterization as well as related information from transcriptome analyses. This initial list of "missing" proteins that will guide the selection of appropriate samples for discovery studies as well as antibody reagents. Also we have illustrated the significant diversity of protein variants (including post-translational modifications, PTMs) using regions on chromosome 17 that contain important oncogenes. We emphasize the need for mandated deposition of proteomics data in public databases, the further development of improved PTM, ASV, and single nucleotide variant (SNV) databases, and the construction of Web sites that can integrate and regularly update such information. In addition, we describe the distribution of both clustered and scattered sets of protein families on the chromosome. Since chromosome 17 is rich in cancer-associated genes, we have focused the clustering of cancer-associated genes in such genomic regions and have used the ERBB2 amplicon as an example of the value of a proteogenomic approach in which one integrates transcriptomic with proteomic information and captures evidence of coexpression through coordinated regulation.

We generated draft genome sequences for two cold-adapted Archaea, Methanogenium frigidum and Methanococcoides burtonii, to identify genotypic characteristics that distinguish them from Archaea with a higher optimal growth temperature (OGT). Comparative genomics revealed trends in amino acid and tRNA composition, and structural features of proteins. Proteins from the cold-adapted Archaea are characterized by a higher content of noncharged polar amino acids, particularly Gln and Thr and a lower content of hydrophobic amino acids, particularly Leu. Sequence data from nine methanogen genomes (OGT 15°–98°C) were used to generate 1111 modeled protein structures. Analysis of the models from the cold-adapted Archaea showed a strong tendency in the solvent-accessible area for more Gln, Thr, and hydrophobic residues and fewer charged residues. A cold shock domain (CSD) protein (CspA homolog) was identified in M. frigidum, two hypothetical proteins with CSD-folds in M. burtonii, and a unique winged helix DNA-binding domain protein in M. burtonii. This suggests that these types of nucleic acid binding proteins have a critical role in cold-adapted Archaea. Structural analysis of tRNA sequences from the Archaea indicated that GC content is the major factor influencing tRNA stability in hyperthermophiles, but not in the psychrophiles, mesophiles or moderate thermophiles. Below an OGT of 60°C, the GC content in tRNA was largely unchanged, indicating that any requirement for flexibility of tRNA in psychrophiles is mediated by other means. This is the first time that comparisons have been performed with genome data from Archaea spanning the growth temperature extremes from psychrophiles to hyperthermophiles.