Genetic diseases are driven by aberrations of the human genome. Identification of such aberrations including structural variations (SVs) is key to our understanding. Conventional short-reads whole ...genome sequencing (cWGS) can identify SVs to base-pair resolution, but utilizes only short-range information and suffers from high false discovery rate (FDR). Linked-reads sequencing (10XWGS) utilizes long-range information by linkage of short-reads originating from the same large DNA molecule. This can mitigate alignment-based artefacts especially in repetitive regions and should enable better prediction of SVs. However, an unbiased evaluation of this technology is not available. In this study, we performed a comprehensive analysis of different types and sizes of SVs predicted by both the technologies and validated with an independent PCR based approach. The SVs commonly identified by both the technologies were highly specific, while validation rate dropped for uncommon events. A particularly high FDR was observed for SVs only found by 10XWGS. To improve FDR and sensitivity, statistical models for both the technologies were trained. Using our approach, we characterized SVs from the MCF7 cell line and a primary breast cancer tumor with high precision. This approach improves SV prediction and can therefore help in understanding the underlying genetics in various diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Multiple genetic events and subsequent clonal evolution drive carcinogenesis, making disease elimination with single-targeted drugs difficult. The multiplicity of gene mutations derived from clonal ...heterogeneity therefore represents an ideal setting for multiepitope tumor vaccination. Here, we used next generation sequencing exome resequencing to identify 962 nonsynonymous somatic point mutations in B16F10 murine melanoma cells, with 563 of those mutations in expressed genes. Potential driver mutations occurred in classical tumor suppressor genes and genes involved in proto-oncogenic signaling pathways that control cell proliferation, adhesion, migration, and apoptosis. Aim1 and Trrap mutations known to be altered in human melanoma were included among those found. The immunogenicity and specificity of 50 validated mutations was determined by immunizing mice with long peptides encoding the mutated epitopes. One-third of these peptides were found to be immunogenic, with 60% in this group eliciting immune responses directed preferentially against the mutated sequence as compared with the wild-type sequence. In tumor transplant models, peptide immunization conferred in vivo tumor control in protective and therapeutic settings, thereby qualifying mutated epitopes that include single amino acid substitutions as effective vaccines. Together, our findings provide a comprehensive picture of the mutanome of B16F10 melanoma which is used widely in immunotherapy studies. In addition, they offer insight into the extent of the immunogenicity of nonsynonymous base substitution mutations. Lastly, they argue that the use of deep sequencing to systematically analyze immunogenicity mutations may pave the way for individualized immunotherapy of cancer patients.
Tumor models are critical for our understanding of cancer and the development of cancer therapeutics. Here, we present an integrated map of the genome, transcriptome and immunome of an epithelial ...mouse tumor, the CT26 colon carcinoma cell line.
We found that Kras is homozygously mutated at p.G12D, Apc and Tp53 are not mutated, and Cdkn2a is homozygously deleted. Proliferation and stem-cell markers, including Top2a, Birc5 (Survivin), Cldn6 and Mki67, are highly expressed while differentiation and top-crypt markers Muc2, Ms4a8a (MS4A8B) and Epcam are not. Myc, Trp53 (tp53), Mdm2, Hif1a, and Nras are highly expressed while Egfr and Flt1 are not. MHC class I but not MHC class II is expressed. Several known cancer-testis antigens are expressed, including Atad2, Cep55, and Pbk. The highest expressed gene is a mutated form of the mouse tumor antigen gp70. Of the 1,688 non-synonymous point variations, 154 are both in expressed genes and in peptides predicted to bind MHC and thus potential targets for immunotherapy development. Based on its molecular signature, we predicted that CT26 is refractory to anti-EGFR mAbs and sensitive to MEK and MET inhibitors, as have been previously reported.
CT26 cells share molecular features with aggressive, undifferentiated, refractory human colorectal carcinoma cells. As CT26 is one of the most extensively used syngeneic mouse tumor models, our data provide a map for the rationale design of mode-of-action studies for pre-clinical evaluation of targeted- and immunotherapies.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Next generation sequencing (NGS) has enabled high throughput discovery of somatic mutations. Detection depends on experimental design, lab platforms, parameters and analysis algorithms. However, ...NGS-based somatic mutation detection is prone to erroneous calls, with reported validation rates near 54% and congruence between algorithms less than 50%. Here, we developed an algorithm to assign a single statistic, a false discovery rate (FDR), to each somatic mutation identified by NGS. This FDR confidence value accurately discriminates true mutations from erroneous calls. Using sequencing data generated from triplicate exome profiling of C57BL/6 mice and B16-F10 melanoma cells, we used the existing algorithms GATK, SAMtools and SomaticSNiPer to identify somatic mutations. For each identified mutation, our algorithm assigned an FDR. We selected 139 mutations for validation, including 50 somatic mutations assigned a low FDR (high confidence) and 44 mutations assigned a high FDR (low confidence). All of the high confidence somatic mutations validated (50 of 50), none of the 44 low confidence somatic mutations validated, and 15 of 45 mutations with an intermediate FDR validated. Furthermore, the assignment of a single FDR to individual mutations enables statistical comparisons of lab and computation methodologies, including ROC curves and AUC metrics. Using the HiSeq 2000, single end 50 nt reads from replicates generate the highest confidence somatic mutation call set.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Coevolution of ticks and the vertebrate immune system has led to the development of immunosuppressive molecules that prevent immediate response of skin-resident immune cells to quickly fend off the ...parasite. In this article, we demonstrate that the tick-derived immunosuppressor sialostatin L restrains IL-9 production by mast cells, whereas degranulation and IL-6 expression are both unaffected. In addition, the expression of IL-1β and IRF4 is strongly reduced in the presence of sialostatin L. Correspondingly, IRF4- or IL-1R-deficient mast cells exhibit a strong impairment in IL-9 production, demonstrating the importance of IRF4 and IL-1 in the regulation of the Il9 locus in mast cells. Furthermore, IRF4 binds to the promoters of Il1b and Il9, suggesting that sialostatin L suppresses mast cell-derived IL-9 preferentially by inhibiting IRF4. In an experimental asthma model, mast cell-specific deficiency in IRF4 or administration of sialostatin L results in a strong reduction in asthma symptoms, demonstrating the immunosuppressive potency of tick-derived molecules.
Background: Aspirin-exacerbated respiratory disease (AERD) is a syndrome characterised by chronic rhinosinusitis, nasal polyps, asthma and aspirin intolerance. An imbalance of eicosanoid metabolism ...with anover-production of cysteinyl leukotrienes (CysLTs) has been associated with AERD. However, the precise mechanisms underlying AERD are unknown. Objective: To establish the transcriptome of the nasal polyp airway epithelial cells derived from AERD patients to discover gene expression patterns in this disease. Methods: Nasal airway epithelial cells were isolated from 12 AERD polyps and 8 AERD non-polyp nasal mucosa samples as controls from the same subjects. Utilising the Illumina HiSeq 2500 platform, RNA samples were sequenced. Potential gene candidate DMRT3 was selected from the differentially-expressed genes for validation. Results: Comparative transcriptome profiling of nasal epithelial cells was accomplished in AERD. A total of 20 genes had twofold mean regulation expression differences or greater. In addition, 8 genes were upregulated, including doublesex and mab-3 related transcription factor 3 (DMRT3), and 12 genes were downregulated. Differentially regulated genes comprised roles in inflammation, defence and immunity. Metabolic process and embryonic development pathways were significantly enriched. Enzyme-linked immune sorbent assay (ELISA) results of DMRT3 in AERD patients were significantly upregulated compared to controls (p = 0.03). Immunohistochemistry (IHC) of AERD nasal polyps localised DMRT3 and was predominantly released in the airway epithelia. Conclusion: Findings suggest that DMRT3 could be potentially involved in nasal polyp development in AERD patients. Furthermore, several genes are downregulated, hinting at the dedifferentiation phenomenon in AERD polyps. However, further studies are imperative to confirm the exact mechanism of polyp formation in AERD patients.
Cortactin is a filamentous actin (F-actin)-binding protein that regulates cytoskeletal dynamics by activating the Arp2/3 complex; it binds to F-actin by means of six N-terminal “cortactin repeats”. ...Gene amplification of 11q13 and consequent overexpression of cortactin in several human cancers is associated with lymph node metastasis. Overexpression as well as tyrosine phosphorylation of cortactin has been reported to enhance cell migration, invasion, and metastasis. Here we report the identification of two alternative splice variants (SV1 and SV2) that affect the cortactin repeats: SV1-cortactin lacks the 6th repeat (exon 11), whereas SV2-cortactin lacks the 5th and 6th repeats (exons 10 and 11). SV-1 cortactin is found co-expressed with wild type (wt)-cortactin in all tissues and cell lines examined, whereas the SV2 isoform is much less abundant. SV1-cortactin binds F-actin and promotes Arp2/3-mediated actin polymerization equally well as wt-cortactin, whereas SV2-cortactin shows reduced F-actin binding and polymerization. Alternative splicing of cortactin does not affect its subcellular localization or growth factor-induced tyrosine phosphorylation. However, cells that overexpress SV1- or SV2-cortactin show significantly reduced cell migration when compared with wt-cortactin-overexpressing cells. Thus, in addition to overexpression and tyrosine phosphorylation, alternative splicing of the F-actin binding domain of cortactin is a new mechanism by which cortactin influences cell migration.
We present a method, seq2HLA, for obtaining an individual's human leukocyte antigen (HLA) class I and II type and expression using standard next generation sequencing RNA-Seq data. RNA-Seq reads are ...mapped against a reference database of HLA alleles, and HLA type, confidence score and locus-specific expression level are determined. We successfully applied seq2HLA to 50 individuals included in the HapMap project, yielding 100% specificity and 94% sensitivity at a P-value of 0.1 for two-digit HLA types. We determined HLA type and expression for previously un-typed Illumina Body Map tissues and a cohort of Korean patients with lung cancer. Because the algorithm uses standard RNA-Seq reads and requires no change to laboratory protocols, it can be used for both existing datasets and future studies, thus adding a new dimension for HLA typing and biomarker studies.
The transcription of tumor mutations from DNA into RNA has implications for biology, epigenetics and clinical practice. It is not clear if mutations are in general transcribed and, if so, at what ...proportion to the wild-type allele. Here, we examined the correlation between DNA mutation allele frequency and RNA mutation allele frequency. We sequenced the exome and transcriptome of tumor cell lines with large copy number variations, identified heterozygous single nucleotide mutations and absolute DNA copy number, and determined the corresponding DNA and RNA mutation allele fraction. We found that 99% of the DNA mutations in expressed genes are expressed as RNA. Moreover, we found a high correlation between the DNA and RNA mutation allele frequency. Exceptions are mutations that cause premature termination codons and therefore activate nonsense-mediated decay. Beyond this, we did not find evidence of any wide-scale mechanism, such as allele-specific epigenetic silencing, preferentially promoting mutated or wild-type alleles. In conclusion, our data strongly suggest that genes are equally transcribed from all alleles, mutated and wild-type, and thus transcribed in proportion to their DNA allele frequency.
We have developed a laboratory information management system (LIMS) for a next-generation sequencing (NGS) laboratory within the existing Galaxy platform. The system provides lab technicians standard ...and customizable sample information forms, barcoded submission forms, tracking of input sample quality, multiplex-capable automatic flow cell design and automatically generated sample sheets to aid physical flow cell preparation. In addition, the platform provides the researcher with a user-friendly interface to create a request, submit accompanying samples, upload sample quality measurements and access to the sequencing results. As the LIMS is within the Galaxy platform, the researcher has access to all Galaxy analysis tools and workflows. The system reports requests and associated information to a message queuing system, such that information can be posted and stored in external systems, such as a wiki. Through an API, raw sequencing results can be automatically pre-processed and uploaded to the appropriate request folder. Developed for the Illumina HiSeq 2000 instrument, many features are directly applicable to other instruments.
The code and documentation are available at http://tron-mainz.de/tron-facilities/computational-medicine/galaxy-lims/
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