Screening the human exome: a comparison of whole genome and whole transcriptome sequencing

Prime editor (PE) has been recently developed to induce efficient and precise on-target editing, whereas its guide RNA (gRNA)-independent off-target effects remain unknown. Here, we used whole-genome and whole-transcriptome sequencing to determine gRNA-independent off-target mutations in cells expanded from single colonies, in which PE generated precise editing at on-target sites. We found that PE triggered no observable gRNA-independent off-target mutation genome-wide or transcriptome-wide in transfected human cells, highlighting its high specificity.

Download Full-text

Tumor profiling from whole-genome and whole transcriptome sequencing to uncover gene fusions and structural variations in clinically relevant cancer genes.

Journal of Clinical Oncology ◽

10.1200/jco.2017.35.15_suppl.e23118 ◽

2017 ◽

Vol 35 (15_suppl) ◽

pp. e23118-e23118

Author(s):

Alexandra E Gylfe ◽

Eve Shinbrot ◽

Boyko Kakaradov ◽

Wayne Delport ◽

Corine K Lau ◽

...

Keyword(s):

Ovarian Cancer ◽

Transcriptome Sequencing ◽

Whole Genome ◽

Gene Fusions ◽

Cancer Genes ◽

Rna Seq ◽

Structural Variations ◽

Whole Transcriptome Sequencing ◽

Somatic Alterations ◽

Whole Transcriptome

e23118 Background: Current targeted cancer therapies rely on the identification of clinically relevant somatic alterations in the tumor. Hotspot gene-panels and exome sequencing are designed to quickly assess somatic variations in frequently mutated regions and/or the coding regions of relevant genes, but they have limited ability to detect complex genomic rearrangements or novel structural variations. Here, we describe an integrative and comprehensive approach to fully characterize the genomic complexity of solid tumors using high throughput whole genome sequencing (WGS) and whole transcriptome sequencing (RNA Seq). Methods: We performed WGS and high-depth sequencing of known cancer genes in 14 paired tumor-normal samples of a variety of tumor types. Tumor-specific somatic alteration assessments included protein-coding mutations, copy number variations, gene fusions and structural variants. In addition, RNA Seq data was analyzed to identify expressed somatic alterations. Results: We identified 2 novel fusion genes as well as important structural alterations which could have clinical and therapeutic implications. We described a novel BRAF fusion gene in a cholangiocarcinoma devoid of other known driver mutations. BRAF fusions have not been described previously in cholangiocarcinoma; this fusion may represent an alternative mechanism for MAPK activation and could be a useful drug target. We also identified a novel NTRK3 fusion partner in a glioblastoma tumor. This fusion may imply a novel mechanism for NTRK3 activation. Finally, we identified numerous tandem duplications in an ovarian cancer. Recent advances describe tandem duplication hotspots in ovarian cancer as a potential driver mechanism characterizing a specific mutational signature. Conclusions: Comprehensive genomics assessment of paired tumor-normal samples through whole-genome and transcriptome sequencing can yield additional clinically actionable genomic characteristics that may not be detected in whole-exome or hotspot gene-panel sequencing. These findings have the potential to aid in clinical decision making.

Download Full-text

Comprehensive Analysis of MYC Translocations in Multiple Myeloma By Whole Genome Sequencing and Whole Transcriptome Sequencing

Blood ◽

10.1182/blood-2019-124704 ◽

2019 ◽

Vol 134 (Supplement_1) ◽

pp. 1774-1774 ◽

Cited By ~ 1

Author(s):

Sonja Seliger ◽

Verena Geirhos ◽

Torsten Haferlach ◽

Wolfgang Kern ◽

Wencke Walter ◽

...

Keyword(s):

Gene Expression ◽

Multiple Myeloma ◽

Genome Sequencing ◽

Transcriptome Sequencing ◽

Comprehensive Analysis ◽

Whole Genome ◽

Employment Equity ◽

Whole Transcriptome Sequencing ◽

Equity Ownership ◽

Whole Transcriptome

Background 8q24 translocations leading to overexpression of MYC are an established prognostic marker in multiple myeloma (MM). Currently FISH (fluorescence in situ hybridization) on CD138+ enriched cell population is the standard diagnostic approach to evaluate the presence of 8q24 translocations. Due to the heterogeneity of breakpoints and technical issues the design of FISH probes is challenging and so far no single FISH assay is capable of detecting each translocation. Aims (1) Evaluation of the frequency of 8q24 translocations in MM by whole genome sequencing (WGS). (2) Determination of the breakpoints on 8q24 and partners. (3) Correlation of WGS data with FISH and MYC expression determined by whole transcriptome sequencing (WTS). Patient cohort and methods CD138+ cell fractions were selected by MACS from bone marrow aspirate samples of 264 patients diagnosed with MM. FISH, WGS and WTS were performed in all cases. For WGS, 151bp paired-end sequences where generated on NovaSeq 6000 machines (Illumina, San Diego, CA). All reported p-values are two-sided and were considered significant at p<0.05. For gene expression (GE) analysis by WTS, estimated gene counts were normalized and the resulting log2 counts per million were used as a proxy of gene expression in each sample. For artefact exclusion, structural variants were checked against 4386 cases covering the spectrum of hematological malignancies. Results In 91/264 (34%) of cases, at least one rearrangement involving the MYC locus (MYCr) was detected by WGS. In 18 of these samples (20%), >1 MYCr was present (114 MYCr in total). Out of these 91 patients, in 32 (35%) the MYCr had been identified by FISH, in 46 cases (51%) it was not detected due to the heterogeneity of breakpoints, while in 13 (14%) patients FISH could not be evaluated (e.g. due to insufficient patient material). Of the 114 MYCr encountered in WGS, 42 involved one of the immunoglobulin loci (IGH n=25, IGK n=9, IGL n=8). The remaining 72 MYCr involved other rare partners. In 29 of these rearrangements, as well as in four complex rearrangements involving IGH or IGK, recurrent rare partners were identified, comprising 1p12/FAM46C (n=6), 6p24.3/BMP6 (n=10), 6q21/FOXO3 (n=4), 7p21.3 (n=3), 11q13/CCND1 (n=5), 20q11.22 (n=5). 43 MYCr involved non-recurrent (single) rare partners, for 4 of these a MYCr was also detected by FISH. The MYCr detected were rather complex: only 34 (30%) showed a simple reciprocal translocation (IGH n=7, IGL n=2, IGK n=4, rare partners n=21), 60 (53%) showed more complex rearrangements (IGH n=12, IGL n=4, IGK n=2, rare partners n=42) and in 20 cases (18%) at least one additional chromosome was involved (IGH n=6, IGL n=3, IGK n=2, rare partners n=9). In 80% of MYCr, breakpoints were located between genomic positions 128.203.605 and 129.375.490 encompassing the pre-described MYC surrounding locus PVT1. IGH-MYC rearrangements showed a tendency to cluster towards the centromere. MYCr involving rare partners showed the broadest breakpoint spectrum and clustered in both directions of the hotspot (Fig 1A). Regarding expression of MYC, all cases showed an overexpression (median GE: 6.9 vs 4.5 in normal controls). Median GE was similar in cases with Ig partners (IGH: 7.1, IGL: 6.7, IGK: 6.6) and non Ig partners (6.8) and also in cases with MYCr detected by FISH (7.0) and cases in which it was not detected by FISH (6.5). Analysis of additional chromosomal aberrations revealed that hyperdiploidy was significantly more frequent in MYCr (n=68/91, 75% vs n=76/173, 44%; p=0.001), while t(11;14) was found significantly less frequent (n=11/91, 12% vs n=49/173, 28%; p=0.003) (Fig 1B). No associations were found between MYCr and other frequent chromosomal abnormalities. Furthermore, molecular mutations frequently occurring in MM (ATM, BRAF, KRAS, NRAS, TP53, IRF4) were analyzed, revealing that patients with MYCr were significantly less frequently associated with mutations in the IRF4 gene (MYCr patients n=1/91; non-MYCr patients n=13/173; p =0.028) (Fig 1C). Conclusions (1) WGS detects ~3x more MYCr compared to FISH. (2) The complexity on the genomic level of MYCr is high, therefore the detection with targeted assays is limited while WGS allows a more comprehensive analysis. (3) MYC expression in cases with MYCr with non Ig partners is comparably high as for Ig-MYC translocations. (4) MYCr are associated with hyperdiploidy, whereas t(11;14) and IRF4 mutations were detected at a lower frequency. Disclosures Seliger: MLL Munich Leukemia Laboratory: Employment. Geirhos:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Baer:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Download Full-text

Mammary-type Myofibroblastoma with Leiomyomatous Differentiation: A Rare Variant with Potential Pitfalls

International Journal of Surgical Pathology ◽

10.1177/10668969211031309 ◽

2021 ◽

pp. 106689692110313

Author(s):

Alexander M. Strait ◽

Julia A. Bridge ◽

Anthony J. Iafrate ◽

Marilyn M. Li ◽

Feng Xu ◽

...

Keyword(s):

Adipose Tissue ◽

Transcriptome Sequencing ◽

Smooth Muscle Actin ◽

The Body ◽

Muscle Actin ◽

Mature Adipose Tissue ◽

Spindle Cells ◽

Whole Transcriptome Sequencing ◽

Whole Transcriptome

Myofibroblastoma is a rare, benign stromal tumor with a diverse morphologic spectrum. Mammary-type myofibroblastoma (MTMF) is the extra-mammary counterpart of this neoplasm and its occurrence throughout the body has become increasingly recognized. Similar morphologic variations of MTMF have now been described which mirror those seen in the breast. We describe a case of intra-abdominal MTMF composed of short fascicles of eosinophilic spindle cells admixed with mature adipose tissue. The spindle cells stained diffusely positive for CD34, desmin, smooth muscle actin, and h-caldesmon by immunohistochemistry. Concurrent loss of RB1 (13q14) and 13q34 loci were confirmed by fluorescence in situ hybridization whereas anchored multiplex PCR and whole transcriptome sequencing did not reveal any pathognomonic fusions suggesting an alternative diagnosis. To the best of our knowledge this is the first documented case of leiomyomatous variant of MTMF.

Download Full-text

The prognosis of gastric adenocarcinoma depends on the crosstalk between immune profiles and tumor genomic alterations.

Journal of Clinical Oncology ◽

10.1200/jco.2021.39.15_suppl.4035 ◽

2021 ◽

Vol 39 (15_suppl) ◽

pp. 4035-4035

Author(s):

Deqiang Wang ◽

Xiaofeng Chen ◽

Yaping Xu ◽

Yuange He ◽

Lifeng Li ◽

...

Keyword(s):

Gastric Adenocarcinoma ◽

Transcriptome Sequencing ◽

Clonal Expansion ◽

Genomic Alterations ◽

M1 Macrophages ◽

Mutual Correlation ◽

Cell Counts ◽

M1 Macrophage ◽

Whole Transcriptome Sequencing ◽

Whole Transcriptome

4035 Background: Gastric adenocarcinoma (GAC) is with a complex microenvironment of tumor cells. A better understanding of the immune landscape of GACs may lead to the improved treatment strategies with ICIs. Methods: To determine whether the molecular characteristics can serve in prognostic stratification of GACs, tumor tissue and blood samples were collected from 231 GAC patients. The median follow-up time was 34 months. The TCR profile was determined by TCR-β CDR3 sequencing while mutation and gene expression profiles were determined by whole exon and whole transcriptome sequencing, respectively. Tumour-infiltrating immune cells were characterized using immunofluorescence (IF) staining. Results: The results showed the OS of patients with high levels of TCR clonality (TCR clonal expansion) was significantly improved compared with patients with low levels (HR = 1.80 and 2.22, p = 0.022 and 0.008, respectively) in the whole group and in the subgroup of patients with stages IB to III disease. Furthermore, low local clonality was an independent risk factor for OS (adjusted-HR = 1.68 and 1.95, p = 0.049 and 0.029, respectively). Thus, TCR clonal expansion in tumour tissue had a strong prognostic value for GAC patients, independent of clinicopathological factors. Based on whole exon and whole transcriptome sequencing, RNF43/FBXW7/ARID2 mutations and local TCR clonality jointly impacted prognosis (p < 0.001), and functional changes in corresponding Wnt pathway/Notch pathway/SWI/SNF complex characterized a GAC subset with enhanced tumour immunogenicity and TCR clonal expansion. TCR CDR3 sequence similarity comparisons yielded clusters of TCR clones of likely similar functions. The most expansive TCR clusters negatively correlated with the percentage of subclonal mutations (Pearson r = -0.8183, p < 0.001), indicating that tumors with less genomic heterogeneity might induce a greater immune response. By IF staining and mutual correlation analysis, only M1 macrophages showed a significant positive correlation with local TCR clonality for epithelia, stroma, and total cell counts. Tumors were categorized according to the density of M1 macrophages, M1 macrophage infiltrated subtype was associated with favorable OS (p = 0.040 and 0.043) and its combination with the local TCR clonality improved prognosis stratification (p < 0.001). Finally, the scoring by local TCR clonality, RNF43/FBXW7/ARID2 mutations and M1 infiltration determined the best prognosis (p < 0.001). Conclusions: TCR profiles were associated with genomic alterations and may serve as a prognostic biomarker for GACs. A multi-omic model including TCR profiles might produce an improved stratification for treatments and outcomes.

Download Full-text

PD57-01 WHOLE-TRANSCRIPTOME SEQUENCING IDENTIFIED GENE EXPRESSION SIGNATURES ASSOCIATED WITH AGGRESSIVE CLEAR CELL RENAL CELL CARCINOMA

The Journal of Urology ◽

10.1016/j.juro.2018.02.2645 ◽

2018 ◽

Vol 199 (4S) ◽

Author(s):

Ken Batai ◽

Elliot Imler ◽

Michael Phung ◽

Robert Bell ◽

Aye Lwin ◽

...

Keyword(s):

Gene Expression ◽

Renal Cell Carcinoma ◽

Cell Carcinoma ◽

Renal Cell ◽

Transcriptome Sequencing ◽

Clear Cell ◽

Cell Renal Cell Carcinoma ◽

Gene Expression Signatures ◽

Whole Transcriptome Sequencing ◽

Whole Transcriptome

Download Full-text

Analysis of mRNAs and ncRNAs and Prediction Competing Endogenous RNA Networks in Colorectal Cancer Chemo-Resistance by Whole-Transcriptome Sequencing

10.21203/rs.3.rs-38686/v1 ◽

2020 ◽

Author(s):

Fei Yao ◽

Chuanren Zhou ◽

Qiyou Huang ◽

Xiaoying Huang ◽

Jie Chen ◽

...

Keyword(s):

Colorectal Cancer ◽

Drug Resistance ◽

Cell Lines ◽

Transcriptome Sequencing ◽

Regulatory Networks ◽

Parental Cell ◽

Resistant Cell ◽

Competing Endogenous Rna ◽

Whole Transcriptome Sequencing ◽

Whole Transcriptome

Abstract Background: Chemo-resistance is a major clinical obstacle to the treatment of colorectal cancer (CRC), mRNAs and non-coding RNAs (ncRNAs) have been reported to modulate the development of chemo-resistance. However, the profiles of mRNAs and ncRNAs as well as competing endogenous RNA (ceRNA) networks in CRC chemo-resistance are still unclear, and whether different drug resistance of CRC have the same mechanisms also needs to be explored. This study aims to uncover the expression of mRNAs and ncRNAs in parental cell lines and different chemo-resistant cell lines, and construct ceRNA regulatory networks by whole-transcriptome sequencing.Methods: The expression of mRNAs and ncRNAs in parental cell lines and drug-resistant cell lines were identified by whole-transcriptome sequencing and bioinformatics methods.Results: A total of 1779 mRNAs, 64 miRNAs, 11 circRNAs and 295 lncRNAs were common differentially expressed in two different chemo-resistant cell lines when compared with the control. In addition, 5,767 lncRNA-miRNA-mRNA relationship pairs and 47 circRNA-miRNA-mRNA pathways were constructed according to ceRNA regulatory rules, in which AC109322.2-hsa-miR-371a-5p-BTNL3 and hsacirc_027876-hsa-miR-582-3p-FREM1 were identified as the most potential ceRNA networks involved in drug resistance to CRC. Gene ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis of two ceRNA regulatory networks showed that the TNF signaling pathway may be crucial in the process of CRC drug resistance.Conclusions: A large number of mRNAs and ncRNAs in chemo-resistant cell lines were different expressed, which may play pivotal roles in development of drug resistance through the ceRNA regulatory network. This study may improve our understanding of the underlying mechanisms and provide a promising therapeutic strategy for CRC chemo-resistance.

Download Full-text