Project description:In-depth information regarding the DFCI OncoPanel sequencing panel has been described previously. Briefly, sequencing is performed using an Illumina HiSeq 2500 system (RRID:SCR_016383) with 2×100 paired-end reads. Samples must meet an average 50X coverage and minimum of 30X coverage for 80% of targets for analysis. For all samples, a board-certified molecular pathologist evaluates alterations and writes interpretations of the results. The three generations of OncoPanel use Mutect (RRID:SCR_000559) and GATK (RRID:SCR_001876) to interrogate possible alterations in the complete exonic DNA sequences of 275, 309, and 447 cancer-related genes, respectively, including substitutions, insertions, and deletions. Because tumor tissues are tested without a paired normal from individual patients, additional informatics steps are taken to identify common single nucleotide polymorphisms (SNPs). Any SNP present at >0.1% in Exome Variant Server, NHLBI GO Exome Sequencing Project (ESP) (RRID:SCR_012761), or present in dbSNP (RRID:SCR_002338) is filtered; however, variants also present in at least twice in COSMIC (RRID:SCR_002260) are rescued for manual review. Variants that appear two or more times in a panel of 150 normal samples sequenced in-house and are not present in COSMIC are also filtered. For copy number alteration (CNA) analysis, the workflow employs an in-house algorithm (RobustCNV) to cover exonic regions of targeted genes as well as select intronic regions. The depth of coverage of these regions is determined by counting the number of reads aligning within defined genomic intervals and then normalized against a panel of normals and corrected for GC bias. The copy number for a segmented genomic interval is calculated as a log2 ratio of the depth of coverage of this sample compared to a panel of normal (non-cancer) samples that are run on the same plate. In parallel (samples not included), additional cases were previously sequenced via the MSK-IMPACT platform. To ensure maximum overlap between the platforms, only single nucleotide variants (SNVs), dinucleotide/oligonucleotide variants (DNVs/ONVs), insertions/deletions (indels), high copy gains (amplifications), and homozygous deletions from 182 genes shared between all versions of both platforms were evaluated in our analyses involving samples from both platforms. To filter pathogenic and passenger mutations, missense, truncating (nonsense, frameshift, and splice site), and in-frame indel mutations from the 182 genes overlapping between the DFCI OncoPanel and MSK-IMPACT panels were subjected to analysis in the Cancer Genome Interpreter (CGI) suite (RRID:SCR_023752) with cancer type set to “Skin Cutaneous Melanoma (SKCM)”. Known pathogenic mutations, defined as those listed as pathogenic in ClinVar (RRID:SCR_006169), OncoKB (RRID:SCR_014782), and/or CGI databases, were included in downstream analyses. Mutations which had not been previously annotated but were predicted to be pathogenic mutations by CGI were also included. Missense SNV mutations called passengers in CGI were subjected to another round of analysis in the ChasmPlus suite with cancer type set to SKCM. Missense mutations called pathogenic via this approach (p < 0.05; FDR Q < 0.3) were salvaged and included in further analyses. All other mutations were excluded. TERT promoter mutations were evaluated via FATHMM-MKL. Mutations called pathogenic via this tool were included in downstream analyses. Clinical data for these cases is available in a supplementary file from the associated publication. OF NOTE, THE RAW SEQUENCING DATA FROM THESE SAMPLES CANNOT BE MADE PUBLICLY AVAILABLE BECAUSE THE RESEARCH PARTICIPANT CONSENT DOES NOT INCLUDE AUTHORIZATION TO SHARE IDENTIFIABLE DATA.
Project description:BRAF mutant colorectal cancer displays a unmet clinical need with poor overall survival. Here we modeled this cancer in the murine system with Braf activation mutation, Trp53 deletion and Nicd1 expression. The respective alleles are recombined in the intestinal epithelium upon systemic tamoxifen injection. Tumors were occuring in the small intestine and in the colon. We conducted exon sequencing to investigate if other mutations occur in the tumor development.
Project description:Intrahepatic cholangiocarcinoma (iCCA) is a fatal bile duct cancer with dismal prognosis and limited therapeutic options. By performing RNA- and exome sequencing analyses we have discovered a novel fusion event, FGFR2-PPHLN1 (16%), and damaging mutations in the ARAF oncogene (11%). Methods: mRNA and gDNA were exctracted from fresh frozen tumor tissues and corresponding normal tissue (n=8 pairs) from patients with iCCA who underwent surgical resection. RNA-seq was performed using Illumina HiSeq 2500 System with 100 nucleotide single-end reads. One sample and its paired non-tumoral tissue were eliminated from the subsequent analysis because of bad RNa quality. The same 8 paired tumors were also analyzed by whole-exome seq. Submitter confirms there are no patient privacy concerns with these data. This dataset is part of the TransQST collection.
Project description:In this study we performed proteogenomic analysis for 9 cell lines of malignant melanoma. The main objectives of the study were identifying the variants originating from point mutations and analyzing the effect of exome data filtering on the outcome of variant identification.
Project description:This study employed an unbiased forward genetic screen to identify suppressors of the maternal-effect lethal phenotype caused by tost-1 dysfunction in Caenorhabditis elegans. The temperature-sensitive tost-1(xf196 ts) mutant exhibits complete embryonic lethality at the restrictive temperature (25°C), providing a robust screening platform. Following EMS mutagenesis of L4 larvae, we screened F2 progeny for viable individuals at 25°C, indicating suppression of the lethal phenotype. After backcrossing to confirm heritability, we performed whole genome sequencing on 21 suppressor strains to identify causative mutations. Additionally we also sequenced the strain used for the mutagenesis as the reference genome.
Project description:We collected blood samples of two non-obstructive azoospermia patients, and performed whole exome sequencing to explore the causal mutations for male infertility.
Project description:During adipocyte differentiation, significant alternative splicing changes occur in association with the adipogenic process. However, little is known about roles played by splicing factors in this process. We observed that mice deficient for the splicing factor SRSF10 exhibit severely impaired development of subcutaneous white adipose tissue as a result of defects in adipogenic differentiation. To identify splicing events responsible for this, RNA-seq analysis was performed using embryonic fibroblast cells. Several SRSF10-affected splicing events that are implicated in adipogenesis have been identified. Skipping of lipin1 exon 7 is controlled by SRSF10-regulated cis-element located in the constitutive exon 8. The activity of this element depends on the binding of SRSF10 and correlates with the relative abundance of lipin1a mRNA. A series of experiments demonstrated that SRSF10 controls the production of lipin1a and thus promotes adipocyte differentiation. Indeed, lipin1a expression could rescue SRSF10-mediated adipogenic defects. Taken together, our results identify SRSF10 as an essential regulator for adipocyte differentiation and also provide new insights into splicing control by SRSF10 in lipin1 pre-mRNA splicing. RNA-seq for wide type (WT) and SRSF10-deficient (KO) mouse MEF cells
Project description:U87MG is a commonly studied grade IV glioma cell line that has been analyzed in at least 1,700 publications over four decades. In order to comprehensively characterize the genome of this cell line and to serve as a model of broad cancer genome sequencing, we have generated greater than 30x genomic sequence coverage using a novel 50-base mate paired strategy with a 1.4kb mean insert library. A total of 1,014,984,286 mate-end and 120,691,623 single-end two-base encoded reads were generated from five slides. All data were aligned using a custom designed tool called BFAST, allowing optimal color space read alignment and accurate identification of DNA variants. The aligned sequence reads and mate pair information identified 35 interchromosomal translocation events, 1,315 structural variations (>100bp), 191,743 small (<21bp) insertions and deletions (indels), and 2,384,470 single nucleotide variations (SNVs). Among these observations, the known homozygous mutation in PTEN was robustly identified, and genes involved in cell adhesion were overrepresented in the mutated gene list. Data were compared to 219,187 heterozygous single nucleotide polymorphisms assayed by Illumina 1M Duo genotyping array to assess accuracy: 93.83% of all SNPs were reliably detected at filtering thresholds that yield greater than 99.99% sequence accuracy. Protein coding sequences were disrupted predominantly in this cancer cell line due to small indels, large deletions and translocations. In total, 512 genes were homozygously mutated, including 154 by SNVs, 178 by small indels, 145 by large microdeletions and 35 by interchromosomal translocations to reveal a highly mutated cell line genome. Of the small homozygously mutated variants, 8 SNVs and 99 indels were novel events not present in dbSNP. These data demonstrate that routine generation of broad cancer genome sequence is possible outside of genome centers. The sequence analysis of U87MG provides an unparalleled level of mutational resolution compared to any cell line to date. Whole genome sequencing of the U87MG brain cancer cell line using the AB SOLiD3 sequencer and genotyping using the Illumina Human1M-Duov3 DNA Analysis BeadChip
Project description:Splicing factor SRSF10 is known to function as a sequence-specific splicing activator. Here, we used RNA-seq coupled with bioinformatics analysis to identify the extensive splicing network regulated by SRSF10 in chicken cells. We found that SRSF10 promoted both exon inclusion and exclusion. Functionally, many of SRSF10-verified alternative exons are linked to pathways of stress and apoptosis. Importantly, reconstituted SRSF10 in knockout cells recovered wild-type splicing patterns and considerably rescued the stress-related defects. Together, our results provide mechanistic insight into SRSF10-regulated alternative splicing events in vivo and demonstrate that SRSF10 plays a crucial role in cell survival under stress conditions. RNA-seq for wide type (WT) and SRSF10-deficient (KO) chicken DT40 cells