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Background: Malignant peripheral nerve sheath tumors (MPNSTs) can arise from atypical neurofibromas (ANF). Loss of the polycomb repressor complex 2 (PRC2) is a common event. Previous studies on PRC2-regulated genes in MPNST used genetic add-back experiments in highly aneuploid MPNST cell lines which may miss PRC2-regulated genes in NF1 -mutant ANF-like precursor cells. A set of PRC2-regulated genes in human Schwann cells (SCs) has not been defined. We hypothesized that PRC2 loss has direct and indirect effects on gene expression resulting in MPNST, so we sought to identify PRC2-regulated genes in immortalized human Schwann cells (iHSCs)., Methods: We engineered NF1 -deficient iHSCs with loss of function SUZ12 or EED mutations. RNA sequencing revealed 1327 differentially expressed genes to define PRC2-regulated genes. To investigate MPNST pathogenesis, we compared genes in iHSCs to consistent gene expression differences between ANF and MPNSTs. Chromatin immunoprecipitation sequencing was used to further define targets. Methylome and proteomic analyses were performed to further identify enriched pathways., Results: We identified potential PRC2-regulated drivers of MPNST progression. Pathway analysis indicates many upregulated cancer-related pathways. We found transcriptional evidence for activated Notch and Sonic Hedgehog (SHH) signaling in PRC2-deficient iHSCs. Functional studies confirm that Notch signaling is active in MPNST cell lines, patient-derived xenografts, and transient cell models of PRC2 deficiency. A combination of MEK and γ-secretase inhibition shows synergy in MPNST cell lines., Conclusions: We identified PRC2-regulated genes and potential drivers of MPNSTs. Our findings support the Notch pathway as a druggable target in MPNSTs. Our identification of PRC2-regulated genes and pathways could result in more novel therapeutic approaches., (© The Author(s) 2024. Published by Oxford University Press, the Society for Neuro-Oncology and the European Association of Neuro-Oncology.)

Obesity exacerbates tissue degeneration and compromises the integrity and reparative potential of mesenchymal stem/stromal cells (MSCs), but the underlying mechanisms have not been sufficiently elucidated. Mitochondria modulate the viability, plasticity, proliferative capacity, and differentiation potential of MSCs. We hypothesized that alterations in the 5-hydroxymethylcytosine (5hmC) profile of mitochondria-related genes may mediate obesity-driven dysfunction of human adipose-derived MSCs. MSCs were harvested from abdominal subcutaneous fat of obese and age/sex-matched non-obese subjects (n = 5 each). The 5hmC profile and expression of nuclear-encoded mitochondrial genes were examined by hydroxymethylated DNA immunoprecipitation sequencing (h MeDIP-seq) and mRNA-seq, respectively. MSC mitochondrial structure (electron microscopy) and function, metabolomics, proliferation, and neurogenic differentiation were evaluated in vitro, before and after epigenetic modulation. hMeDIP-seq identified 99 peaks of hyper-hydroxymethylation and 150 peaks of hypo-hydroxymethylation in nuclear-encoded mitochondrial genes from Obese- versus Non-obese-MSCs. Integrated hMeDIP-seq/mRNA-seq analysis identified a select group of overlapping (altered levels of both 5hmC and mRNA) nuclear-encoded mitochondrial genes involved in ATP production, redox activity, cell proliferation, migration, fatty acid metabolism, and neuronal development. Furthermore, Obese-MSCs exhibited decreased mitochondrial matrix density, membrane potential, and levels of fatty acid metabolites, increased superoxide production, and impaired neuronal differentiation, which improved with epigenetic modulation. Obesity elicits epigenetic changes in mitochondria-related genes in human adipose-derived MSCs, accompanied by structural and functional changes in their mitochondria and impaired fatty acid metabolism and neurogenic differentiation capacity. These observations may assist in developing novel therapies to preserve the potential of MSCs for tissue repair and regeneration in obese individuals., (© 2024. The Author(s).)

Obesity is an epidemic with myriad health effects, but little is understood regarding individual obese phenotypes and how they may respond to therapy. Epigenetic changes associated with obesity have been detected in blood, liver, pancreas, and adipose tissues. Previous work found that dietary glucose hyperabsorption occurs in some obese subjects, but detailed transcriptional or epigenomic features of the intestine associated with this phenotype are unknown. This study evaluated differentially expressed genes and relative chromatin accessibility in intestinal organoids established from donors classified as lean, obese, or obese hyperabsorptive by body mass index and glucose transport assays. Transcriptomic analysis indicated that obese hyperabsorptive subjects have significantly upregulated dietary nutrient absorption proteins and downregulated type I interferon targets. Chromatin accessibility and transcription factor footprinting suggested that enhanced binding of HNF4G promotes the obese hyperabsorption phenotype. Quantitative PCR assessment in a larger subject cohort suggested that intestinal epithelial expression of CUBN, GIP, and SLC2A5 have high correlation with hyperabsorption. The obese hyperabsorption phenotype is characterized by transcriptional changes that support increased nutrient uptake and may be driven by differentially accessible chromatin. Recognizing unique intestinal phenotypes in obesity provides new perspective in considering therapeutic targets and options to manage the disease.

Lactic acid has emerged as an important modulator of immune cell function. It can be produced by both gut microbiota and the host metabolism at homeostasis and during disease states. The production of lactic acid in the gut microenvironment is vital for tissue homeostasis. In the present study, we examined how lactic acid integrates cellular metabolism to shape the epigenome of macrophages during pro-inflammatory response. We found that lactic acid serves as a primary fuel source to promote histone H3K27 acetylation, which allows the expression of immunosuppressive gene program including Nr4a1. Consequently, macrophage pro-inflammatory function was transcriptionally repressed. Furthermore, the histone acetylation induced by lactic acid promotes a form of long-term immunosuppression ("trained immunosuppression"). Pre-exposure to lactic acid induces lipopolysaccharide tolerance. These findings thus indicate that lactic acid sensing and its effect on chromatin remodeling in macrophages represent a key homeostatic mechanism that can provide a tolerogenic tissue microenvironment., Competing Interests: Declaration of interests The authors declare no competing interests., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Loss-of-function mutations in the genes encoding PINK1 and PRKN result in early-onset Parkinson disease (EOPD). Together the encoded enzymes direct a neuroprotective pathway that ensures the elimination of damaged mitochondria via autophagy. We performed a genome-wide high content imaging miRNA screen for inhibitors of the PINK1-PRKN pathway and identified all three members of the miRNA family 29 (miR-29). Using RNAseq we identified target genes and found that siRNA against ATG9A phenocopied the effects of miR-29 and inhibited the initiation of PINK1-PRKN mitophagy. Furthermore, we discovered two rare, potentially deleterious, missense variants (p.R631W and p.S828L) in our EOPD cohort and tested them experimentally in cells. While expression of wild-type ATG9A was able to rescue the effects of miR-29a, the EOPD-associated variants behaved like loss-of-function mutations. Together, our study validates miR-29 and its target gene ATG9A as novel regulators of mitophagy initiation. It further serves as proof-of-concept of finding novel, potentially disease-causing EOPD-linked variants specifically in mitophagy regulating genes. The nomination of genetic variants and biological pathways is important for the stratification and treatment of patients that suffer from devastating diseases, such as EOPD., Competing Interests: COMPETING INTERESTS Mayo Clinic, FCF and WS have filed a patent related to PRKN activators. WEG is an employee of Revvity, formerly known as Perkin Elmer (Waltham, MA, USA). ZKW serves as PI or Co-PI on Biohaven Pharmaceuticals, Inc. (BHV4157-206) and Vigil Neuroscience, Inc. (VGL101-01.002, VGL101-01.201, PET tracer development protocol, Csf1r biomarker and repository project, and ultra-high field MRI in the diagnosis and management of CSF1R-related adult-onset leukoencephalopathy with axonal spheroids and pigmented glia) projects/grants. He serves as Co-PI of the Mayo Clinic APDA Center for Advanced Research, as an external advisory board member for the Vigil Neuroscience, Inc., and as a consultant on neurodegenerative medical research for Eli Lilly & Company. All other authors declare they have no competing interests. This research was conducted in compliance with Mayo Clinic conflict of interest policies.

Background & Aims: Although depletion of neuronal nitric oxide synthase (NOS1)-expressing neurons contributes to gastroparesis, stimulating nitrergic signaling is not an effective therapy. We investigated whether hypoxia-inducible factor 1α (HIF1A), which is activated by high O 2 consumption in central neurons, is a Nos1 transcription factor in enteric neurons and whether stabilizing HIF1A reverses gastroparesis., Methods: Mice with streptozotocin-induced diabetes, human and mouse tissues, NOS1 + mouse neuroblastoma cells, and isolated nitrergic neurons were studied. Gastric emptying of solids and volumes were determined by breath test and single-photon emission computed tomography, respectively. Gene expression was analyzed by RNA-sequencing, microarrays, immunoblotting, and immunofluorescence. Epigenetic assays included chromatin immunoprecipitation sequencing (13 targets), chromosome conformation capture sequencing, and reporter assays. Mechanistic studies used Cre-mediated recombination, RNA interference, and clustered regularly interspaced short palindromic repeats (CRISPR)-CRISPR-associated protein 9 (Cas9)-mediated epigenome editing., Results: HIF1A signaling from physiological intracellular hypoxia was active in mouse and human NOS1 + myenteric neurons but reduced in diabetes. Deleting Hif1a in Nos1-expressing neurons reduced NOS1 protein by 50% to 92% and delayed gastric emptying of solids in female but not male mice. Stabilizing HIF1A with roxadustat (FG-4592), which is approved for human use, restored NOS1 and reversed gastroparesis in female diabetic mice. In nitrergic neurons, HIF1A up-regulated Nos1 transcription by binding and activating proximal and distal cis-regulatory elements, including newly discovered super-enhancers, facilitating RNA polymerase loading and pause-release, and by recruiting cohesin to loop anchors to alter chromosome topology., Conclusions: Pharmacologic HIF1A stabilization is a novel, translatable approach to restoring nitrergic signaling and treating diabetic gastroparesis. The newly recognized effects of HIF1A on chromosome topology may provide insights into physioxia- and ischemia-related organ function., (Copyright © 2023 AGA Institute. Published by Elsevier Inc. All rights reserved.)

Background: The development of Crohn's disease [CD] involves immune cell signalling pathways regulated by epigenetic modifications. Aberrant DNA methylation has been identified in peripheral blood and bulk intestinal tissue from CD patients. However, the DNA methylome of disease-associated intestinal CD4+ lymphocytes has not been evaluated., Materials and Methods: Genome-wide DNA methylation sequencing was performed from terminal ileum CD4+ cells from 21 CD patients and 12 age- and sex-matched controls. Data were analysed for differentially methylated CpGs [DMCs] and methylated regions [DMRs]. Integration was performed with RNA-sequencing data to evaluate the functional impact of DNA methylation changes on gene expression. DMRs were overlapped with regions of differentially open chromatin [by ATAC-seq] and CCCTC-binding factor [CTCF] binding sites [by ChIP-seq] between peripherally derived Th17 and Treg cells., Results: CD4+ cells in CD patients had significantly increased DNA methylation compared to those from the controls. A total of 119 051 DMCs and 8113 DMRs were detected. While hypermethylated genes were mostly related to cell metabolism and homeostasis, hypomethylated genes were significantly enriched within the Th17 signalling pathway. The differentially enriched ATAC regions in Th17 cells [compared to Tregs] were hypomethylated in CD patients, suggesting heightened Th17 activity. There was significant overlap between hypomethylated DNA regions and CTCF-associated binding sites., Conclusions: The methylome of CD patients shows an overall dominant hypermethylation yet hypomethylation is more concentrated in proinflammatory pathways, including Th17 differentiation. Hypomethylation of Th17-related genes associated with areas of open chromatin and CTCF binding sites constitutes a hallmark of CD-associated intestinal CD4+ cells., (© The Author(s) 2023. Published by Oxford University Press on behalf of European Crohn’s and Colitis Organisation. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Cellular senescence and circadian dysregulation are biological hallmarks of aging. Whether they are coordinately regulated has not been thoroughly studied. We hypothesize that BMAL1, a pioneer transcription factor and master regulator of the molecular circadian clock, plays a role in the senescence program. Here, we demonstrate BMAL1 is significantly upregulated in senescent cells and has altered rhythmicity compared to non-senescent cells. Through BMAL1-ChIP-seq, we show that BMAL1 is uniquely localized to genomic motifs associated with AP-1 in senescent cells. Integration of BMAL1-ChIP-seq data with RNA-seq data revealed that BMAL1 presence at AP-1 motifs is associated with active transcription. Finally, we showed that BMAL1 contributes to AP-1 transcriptional control of key features of the senescence program, including altered regulation of cell survival pathways, and confers resistance to drug-induced apoptosis. Overall, these results highlight a previously unappreciated role of the core circadian clock component BMAL1 on the molecular phenotype of senescent cells.

Objective: Pancreatic ductal adenocarcinoma (PDAC) displays a remarkable propensity towards therapy resistance. However, molecular epigenetic and transcriptional mechanisms enabling this are poorly understood. In this study, we aimed to identify novel mechanistic approaches to overcome or prevent resistance in PDAC., Design: We used in vitro and in vivo models of resistant PDAC and integrated epigenomic, transcriptomic, nascent RNA and chromatin topology data. We identified a JunD-driven subgroup of enhancers, called interactive hubs (iHUBs), which mediate transcriptional reprogramming and chemoresistance in PDAC., Results: iHUBs display characteristics typical for active enhancers (H3K27ac enrichment) in both therapy sensitive and resistant states but exhibit increased interactions and production of enhancer RNA (eRNA) in the resistant state. Notably, deletion of individual iHUBs was sufficient to decrease transcription of target genes and sensitise resistant cells to chemotherapy. Overlapping motif analysis and transcriptional profiling identified the activator protein 1 (AP1) transcription factor JunD as a master transcription factor of these enhancers. JunD depletion decreased iHUB interaction frequency and transcription of target genes. Moreover, targeting either eRNA production or signaling pathways upstream of iHUB activation using clinically tested small molecule inhibitors decreased eRNA production and interaction frequency, and restored chemotherapy responsiveness in vitro and in vivo. Representative iHUB target genes were found to be more expressed in patients with poor response to chemotherapy compared with responsive patients., Conclusion: Our findings identify an important role for a subgroup of highly connected enhancers (iHUBs) in regulating chemotherapy response and demonstrate targetability in sensitisation to chemotherapy., Competing Interests: Competing interests: None declared., (© Author(s) (or their employer(s)) 2023. No commercial re-use. See rights and permissions. Published by BMJ.)

Vascular dysfunction is a hallmark of chronic diseases in elderly. The contribution of the vasculature to lung repair and fibrosis is not fully understood. Here, we performed an epigenetic and transcriptional analysis of lung endothelial cells (ECs) from young and aged mice during the resolution or progression of bleomycin-induced lung fibrosis. We identified the transcription factor ETS-related gene (ERG) as putative orchestrator of lung capillary homeostasis and repair, and whose function is dysregulated in aging. ERG dysregulation is associated with reduced chromatin accessibility and maladaptive transcriptional responses to injury. Loss of endothelial ERG enhances paracrine fibroblast activation in vitro, and impairs lung fibrosis resolution in young mice in vivo. scRNA-seq of ERG deficient mouse lungs reveales transcriptional and fibrogenic abnormalities resembling those associated with aging and human lung fibrosis, including reduced number of general capillary (gCap) ECs. Our findings demonstrate that lung endothelial chromatin remodeling deteriorates with aging leading to abnormal transcription, vascular dysrepair, and persistent fibrosis following injury., (© 2022. The Author(s).)

Periprosthetic joint infection (PJI), a devastating complication of total joint replacement, is of incompletely understood pathogenesis and may sometimes be challenging to clinically distinguish from other causes of arthroplasty failure. We characterized human gene expression in 93 specimens derived from surfaces of resected arthroplasties, comparing transcriptomes of subjects with infection- versus non-infection-associated arthroplasty failure. Differential gene expression analysis confirmed 28 previously reported potential biomarkers of PJI, including bactericidal/permeability increasing protein (BPI), cathelicidin antimicrobial peptide (CAMP), C-C-motif chemokine ligand 3 (CCL3), 4(CCL4) and C-X-C-motif chemokine ligand 2 (CXCL2), colony stimulating factor 2 receptor beta (CSF2RB), colony stimulating factor 3 (CSF3), alpha-defensin (DEFA4), Fc fragment of IgG receptor 1B (CD64B), intercellular adhesion molecule 1 (ICAM1), interferon gamma (IFNG), interleukin 13 receptor subunit alpha 2 (IL13RA2), interleukin 17D (IL17D), interleukin 1 (IL1A, IL1B, IL1RN), interleukin 2 receptors (IL2RA, IL2RG), interleukin 5 receptor (IL5RA), interleukin 6 (IL6), interleukin 8 (IL8), lipopolysaccharide binding protein (LBP), lipocalin (LCN2), lactate dehydrogenase C (LDHC), lactotransferrin (LTF), matrix metallopeptidase 3 (MMP3), peptidase inhibitor 3 (PI3), and vascular endothelial growth factor A (VEGFA), and identified three novel molecules of potential diagnostic use for detection of PJI, namely C-C-motif chemokine ligand CCL20, coagulation factor VII (F7), and B cell receptor FCRL4. Comparative analysis of infections caused by staphylococci versus bacteria other than staphylococci and Staphylococcus aureus versus Staphylococcus epidermidis showed elevated expression of interleukin 13 (IL13), IL17D, and MMP3 in staphylococcal infections, and of IL1B, IL8, and platelet factor PF4V1 in S. aureus compared to S. epidermidis infections. Pathway analysis of over-represented genes suggested activation of host immune response and cellular maintenance and repair functions in response to invasion of infectious agents. The data presented provides new potential targets for diagnosis of PJI and for differentiation of PJI caused by different infectious agents., (Copyright © 2022 The Authors. Published by Elsevier B.V. All rights reserved.)

We explored the feasibility of obtaining accurate HLA type using pre-existing NGS data not generated for HLA purposes. 83 exomes and 500 targeted NGS pharmacogenomic panels were analyzed using Omixon HLA Explore, OptiType, and/or HLA-Genotyper software. Results were compared against clinical HLA genotyping. 765 (94.2%) Omixon and 769 (94.7%) HLA-Genotyper of 812 germline allele calls across class I/II loci and 402 (99.5%) of 404 OptiType class I calls were concordant to the second field (i.e. HLA-A*02:01). An additional 19 (2.3%) Omixon, 39 (4.8%) HLA-Genotyper, and 2 (0.5%) OptiType allele calls were first field concordant (i.e. HLA-A*02). Using Omixon, four alleles (0.4%) were discordant and 24 (3.0%) failed to call, while 4 alleles (0.4%) were discordant using HLA-Genotyper. Tumor exomes were also evaluated and were 85.4%, 91.6%, and 100% concordant (Omixon and HLA-Genotyper with 96 alleles tested, and Optitype with 48 class I alleles, respectively). The 15 exomes and 500 pharmacogenomic panels were 100% concordant for each pharmacogenomic allele tested. This work has broad implications spanning future clinical care (pharmacogenomics, tumor response to immunotherapy, autoimmunity, etc.) and research applications., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 American Society for Histocompatibility and Immunogenetics. Published by Elsevier Inc. All rights reserved.)

Background: Archived formalin fixed paraffin embedded (FFPE) samples are valuable clinical resources to examine clinically relevant morphology features and also to study genetic changes. However, DNA quality and quantity of FFPE samples are often sub-optimal, and resulting NGS-based genetics variant detections are prone to false positives. Evaluations of wet-lab and bioinformatics approaches are needed to optimize variant detection from FFPE samples., Results: As a pilot study, we designed within-subject triplicate samples of DNA derived from paired FFPE and fresh frozen breast tissues to highlight FFPE-specific artifacts. For FFPE samples, we tested two FFPE DNA extraction methods to determine impact of wet-lab procedures on variant calling: QIAGEN QIAamp DNA Mini Kit ("QA"), and QIAGEN GeneRead DNA FFPE Kit ("QGR"). We also used negative-control (NA12891) and positive control samples (Horizon Discovery Reference Standard FFPE). All DNA sample libraries were prepared for NGS according to the QIAseq Human Breast Cancer Targeted DNA Panel protocol and sequenced on the HiSeq 4000. Variant calling and filtering were performed using QIAGEN Gene Globe Data Portal. Detailed variant concordance comparisons and mutational signature analysis were performed to investigate effects of FFPE samples compared to paired fresh frozen samples, along with different DNA extraction methods. In this study, we found that five times or more variants were called with FFPE samples, compared to their paired fresh-frozen tissue samples even after applying molecular barcoding error-correction and default bioinformatics filtering recommended by the vendor. We also found that QGR as an optimized FFPE-DNA extraction approach leads to much fewer discordant variants between paired fresh frozen and FFPE samples. Approximately 92% of the uniquely called FFPE variants were of low allelic frequency range (< 5%), and collectively shared a "C > T|G > A" mutational signature known to be representative of FFPE artifacts resulting from cytosine deamination. Based on control samples and FFPE-frozen replicates, we derived an effective filtering strategy with associated empirical false-discovery estimates., Conclusions: Through this study, we demonstrated feasibility of calling and filtering genetic variants from FFPE tissue samples using a combined strategy with molecular barcodes, optimized DNA extraction, and bioinformatics methods incorporating genomics context such as mutational signature and variant allelic frequency.

With the emergence of RNA sequencing technologies, metatranscriptomic studies are rapidly gaining attention as they simultaneously provide insight into gene expression profiles and therefore disease association pathways of microbial pathogens and their hosts. This approach, therefore, holds promise for applicability in infectious disease diagnostics. A challenge of this approach in the clinical setting is the low amount and quality of RNA, especially microbial RNA in most clinically-infected specimens. Here, we compared two commercially available stranded cDNA library preparation kits, the NuGEN Ovation SoLo RNA-Seq System and the Illumina TruSeq Stranded Total RNA, using RNA extracted from synovial and sonicate fluids from a subject with periprosthetic joint infection. The Ovation SoLo RNA-Seq System provided more useful transcriptomic data for the infecting bacterium, whereas the TruSeq Stranded Total RNA kit provided more useful human transcriptomic data., (Copyright © 2018 Elsevier B.V. All rights reserved.)

Aims: To compare the performance of four sequence-based and one microarray methods for DNA methylation profiling., Methods: DNA from two cell lines were profiled by reduced representation bisulfite sequencing, methyl capture sequencing (SS-Meth Seq), NimbleGen SeqCapEpi CpGiant(Nimblegen MethSeq), methylated DNA immunoprecipitation (MeDIP) and the Human Methylation 450 Bead Chip (Meth450K)., Results & Conclusion: Despite differences in genome-wide coverage, high correlation and concordance were observed between different methods. Significant overlap of differentially methylated regions was identified between sequenced-based platforms. MeDIP provided the best coverage for the whole genome and gene body regions, while RRBS and Nimblegen MethSeq were superior for CpGs in CpG islands and promoters. Methylation analyses can be achieved by any of the five methods but understanding their differences may better address the research question being posed.

Motivation: RNA-seq has become the method of choice to quantify genes and exons, discover novel transcripts and detect fusion genes. However, reliable variant identification from RNA-seq data remains challenging because of the complexities of the transcriptome, the challenges of accurately mapping exon boundary spanning reads and the bias introduced during the sequencing library preparation., Method: We developed RVboost, a novel method specific for RNA variant prioritization. RVboost uses several attributes unique in the process of RNA library preparation, sequencing and RNA-seq data analyses. It uses a boosting method to train a model of 'good quality' variants using common variants from HapMap, and prioritizes and calls the RNA variants based on the trained model. We packaged RVboost in a comprehensive workflow, which integrates tools of variant calling, annotation and filtering., Results: RVboost consistently outperforms the variant quality score recalibration from the Genome Analysis Tool Kit and the RNA-seq variant-calling pipeline SNPiR in 12 RNA-seq samples using ground-truth variants from paired exome sequencing data. Several RNA-seq-specific attributes were identified as critical to differentiate true and false variants, including the distance of the variant positions to exon boundaries, and the percent of the reads supporting the variant in the first six base pairs. The latter identifies false variants introduced by the random hexamer priming during the library construction., Availability and Implementation: The RVboost package is implemented to readily run in Mac or Linux environments. The software and user manual are available at http://bioinformaticstools.mayo.edu/research/rvboost/., (© The Author 2014. Published by Oxford University Press.)

Motivation: Exome sequencing (exome-seq) data, which are typically used for calling exonic mutations, have also been utilized in detecting DNA copy number variations (CNVs). Despite the existence of several CNV detection tools, there is still a great need for a sensitive and an accurate CNV-calling algorithm with built-in QC steps, and does not require a paired reference for each sample., Results: We developed a novel method named PatternCNV, which (i) accounts for the read coverage variations between exons while leveraging the consistencies of this variability across different samples; (ii) reduces alignment BAM files to WIG format and therefore greatly accelerates computation; (iii) incorporates multiple QC measures designed to identify outlier samples and batch effects; and (iv) provides a variety of visualization options including chromosome, gene and exon-level views of CNVs, along with a tabular summarization of the exon-level CNVs. Compared with other CNV-calling algorithms using data from a lymphoma exome-seq study, PatternCNV has higher sensitivity and specificity., Availability and Implementation: The software for PatternCNV is implemented using Perl and R, and can be used in Mac or Linux environments. Software and user manual are available at http://bioinformaticstools.mayo.edu/research/patterncnv/, and R package at https://github.com/topsoil/patternCNV/., (© The Author 2014. Published by Oxford University Press.)

Objectives: The sequencing by the PolyA selection is the most common approach for library preparation. With limited amount or degraded RNA, alternative protocols such as the NuGEN have been developed. However, it is not yet clear how the different library preparations affect the downstream analyses of the broad applications of RNA sequencing., Methods and Materials: Eight human mammary epithelial cell (HMEC) lines with high quality RNA were sequenced by Illumina's mRNA-Seq PolyA selection and NuGEN ENCORE library preparation. The following analyses and comparisons were conducted: 1) the numbers of genes captured by each protocol; 2) the impact of protocols on differentially expressed gene detection between biological replicates; 3) expressed single nucleotide variant (SNV) detection; 4) non-coding RNAs, particularly lincRNA detection; and 5) intragenic gene expression., Results: Sequences from the NuGEN protocol had lower (75%) alignment rate than the PolyA (over 90%). The NuGEN protocol detected fewer genes (12-20% less) with a significant portion of reads mapped to non-coding regions. A large number of genes were differentially detected between the two protocols. About 17-20% of the differentially expressed genes between biological replicates were commonly detected between the two protocols. Significantly higher numbers of SNVs (5-6 times) were detected in the NuGEN samples, which were largely from intragenic and intergenic regions. The NuGEN captured fewer exons (25% less) and had higher base level coverage variance. While 6.3% of reads were mapped to intragenic regions in the PolyA samples, the percentages were much higher (20-25%) for the NuGEN samples. The NuGEN protocol did not detect more known non-coding RNAs such as lincRNAs, but targeted small and "novel" lincRNAs., Conclusion: Different library preparations can have significant impacts on downstream analysis and interpretation of RNA-seq data. The NuGEN provides an alternative for limited or degraded RNA but it has limitations for some RNA-seq applications.