scholarly journals Transcript-targeted analysis reveals isoform alterations and double-hop fusions in breast cancer

2021 ◽  
Vol 4 (1) ◽  
Author(s):  
Shinichi Namba ◽  
Toshihide Ueno ◽  
Shinya Kojima ◽  
Kenya Kobayashi ◽  
Katsushige Kawase ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Biology ◽  
Sequence Data ◽  
Short Form ◽  
Transcriptome Assembly ◽  
Full Length ◽  
Clinical Samples ◽  
Aberrant Expression ◽  
Fusion Transcripts ◽  
Long Read

AbstractAlthough transcriptome alteration is an essential driver of carcinogenesis, the effects of chromosomal structural alterations on the cancer transcriptome are not yet fully understood. Short-read transcript sequencing has prevented researchers from directly exploring full-length transcripts, forcing them to focus on individual splice sites. Here, we develop a pipeline for Multi-Sample long-read Transcriptome Assembly (MuSTA), which enables construction of a transcriptome from long-read sequence data. Using the constructed transcriptome as a reference, we analyze RNA extracted from 22 clinical breast cancer specimens. We identify a comprehensive set of subtype-specific and differentially used isoforms, which extended our knowledge of isoform regulation to unannotated isoforms including a short form TNS3. We also find that the exon–intron structure of fusion transcripts depends on their genomic context, and we identify double-hop fusion transcripts that are transcribed from complex structural rearrangements. For example, a double-hop fusion results in aberrant expression of an endogenous retroviral gene, ERVFRD-1, which is normally expressed exclusively in placenta and is thought to protect fetus from maternal rejection; expression is elevated in several TCGA samples with ERVFRD-1 fusions. Our analyses provide direct evidence that full-length transcript sequencing of clinical samples can add to our understanding of cancer biology and genomics in general.

Multi-sample Full-length Transcriptome Analysis of 22 Breast Cancer Clinical Specimens with Long-Read Sequencing

2020 ◽  
Author(s):  
Shinichi Namba ◽  
Toshihide Ueno ◽  
Shinya Kojima ◽  
Yosuke Tanaka ◽  
Satoshi Inoue ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Cancer Biology ◽  
Transcriptome Assembly ◽  
Simulated Data ◽  
Fusion Transcript ◽  
Full Length ◽  
Aberrant Expression ◽  
Clinical Specimens ◽  
Fusion Transcripts ◽  
Long Read

AbstractAlthough transcriptome alteration is considered as one of the essential drivers of carcinogenesis, conventional short-read RNAseq technology has limited researchers from directly exploring full-length transcripts, only focusing on individual splice sites. We developed a pipeline for Multi-Sample long-read Transcriptome Assembly, MuSTA, and showed through simulations that it enables construction of transcriptome from the transcripts expressed in target samples and more accurate evaluation of transcript usage. We applied it to 22 breast cancer clinical specimens to successfully acquire cohort-wide full-length transcriptome from long-read RNAseq data. By comparing isoform existence and expression between estrogen receptor positive and triple-negative subtypes, we obtained a comprehensive set of subtype-specific isoforms and differentially used isoforms which consisted of both known and unannotated isoforms. We have also found that exon-intron structure of fusion transcripts tends to depend on their genomic regions, and have found three-piece fusion transcripts that were transcribed from complex structural rearrangements. For example, a three-piece fusion transcript resulted in aberrant expression of an endogenous retroviral gene, ERVFRD-1, which is normally expressed exclusively in placenta and supposed to protect fetus from maternal rejection, and expression of which were increased in several TCGA samples with ERVFRD-1 fusions. Our analyses of real clinical specimens and simulated data provide direct evidence that full-length transcript sequencing in multiple samples can add to our understanding of cancer biology and genomics in general.

scholarly journals Identification of a Modified HOXB9 mRNA in Breast Cancer

2020 ◽  
Vol 2020 ◽  
pp. 1-10
Author(s):  
Ayako Nakashoji ◽  
Tetsu Hayashida ◽  
Yuko Kawai ◽  
Masayuki Kikuchi ◽  
Rurina Watanuki ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Human Breast Cancer ◽  
Cancer Cell Line ◽  
Clinical Samples ◽  
The Novel ◽  
Rna Seq ◽  
Human Breast ◽  
Aberrant Expression ◽  
Novel Variant

First identified as a developmental gene, HOXB9 is also known to be involved in tumor biological processes, and its aberrant expression correlates with poor prognosis of various cancers. In this study, we isolated a homeodomain-less, novel HOXB9 variant (HOXB9v) from human breast cancer cell line-derived mRNA. We confirmed that the novel variant was produced from variationless HOXB9 genomic DNA. RT-PCR of mRNA isolated from clinical samples and reanalysis of publicly available RNA-seq data proved that the new transcript is frequently expressed in human breast cancer. Exogenous HOXB9v expression significantly enhanced the proliferation of breast cancer cells, and gene ontology analysis indicated that apoptotic signaling was suppressed in these cells. Considering that HOXB9v lacks key domains of homeobox proteins, its behavior could be completely different from that of the previously described variationless HOXB9. Because none of the previous studies on HOXB9 have considered the presence of HOXB9v, further research analyzing the two transcripts individually is warranted to re-evaluate the true role of HOXB9 in cancer.

scholarly journals A draft genome sequence for the Ixodes scapularis cell line, ISE6

F1000Research ◽  
2018 ◽  
Vol 7 ◽  
pp. 297 ◽  
Author(s):  
Jason R. Miller ◽  
Sergey Koren ◽  
Kari A. Dilley ◽  
Derek M. Harkins ◽  
Timothy B. Stockwell ◽  
...  
Keyword(s):  
Cell Line ◽  
Genome Sequence ◽  
Single Molecule ◽  
Reference Genome ◽  
Sequence Data ◽  
Ixodes Scapularis ◽  
Draft Genome ◽  
Clinical Samples ◽  
Draft Assembly ◽  
Long Read

Background:The tick cell line ISE6, derived fromIxodes scapularis, is commonly used for amplification and detection of arboviruses in environmental or clinical samples.Methods:To assist with sequence-based assays, we sequenced the ISE6 genome with single-molecule, long-read technology.Results:The draft assembly appears near complete based on gene content analysis, though it appears to lack some instances of repeats in this highly repetitive genome. The assembly appears to have separated the haplotypes at many loci. DNA short read pairs, used for validation only, mapped to the cell line assembly at a higher rate than they mapped to theIxodes scapularisreference genome sequence.Conclusions:The assembly could be useful for filtering host genome sequence from sequence data obtained from cells infected with pathogens.

scholarly journals TRIM47 activates NF-κB signaling via PKC-ε/PKD3 stabilization and contributes to endocrine therapy resistance in breast cancer

2021 ◽  
Vol 118 (35) ◽  
pp. e2100784118
Author(s):  
Kotaro Azuma ◽  
Kazuhiro Ikeda ◽  
Takashi Suzuki ◽  
Kenjiro Aogi ◽  
Kuniko Horie-Inoue ◽  
...  
Keyword(s):  
Breast Cancer ◽  
Protein Kinase ◽  
Cancer Patients ◽  
Endocrine Therapy ◽  
Cancer Biology ◽  
Therapy Resistance ◽  
Clinical Samples ◽  
Breast Cancer Patients ◽  
Endocrine Therapy Resistance ◽  
Mcf 7

Increasing attention has been paid to roles of tripartite motif–containing (TRIM) family proteins in cancer biology, often functioning as E3 ubiquitin ligases. In the present study, we focus on a contribution of TRIM47 to breast cancer biology, particularly to endocrine therapy resistance, which is a major clinical problem in breast cancer treatment. We performed immunohistochemical analysis of TRIM47 protein expression in 116 clinical samples of breast cancer patients with postoperative endocrine therapy using tamoxifen. Our clinicopathological study showed that higher immunoreactivity scores of TRIM47 were significantly associated with higher relapse rate of breast cancer patients (P = 0.012). As functional analyses, we manipulated TRIM47 expression in estrogen receptor–positive breast cancer cells MCF-7 and its 4-hydroxytamoxifen (OHT)-resistant derivative OHTR, which was established in a long-term culture with OHT. TRIM47 promoted both MCF-7 and OHTR cell proliferation. MCF-7 cells acquired tamoxifen resistance by overexpressing exogenous TRIM47. We found that TRIM47 enhances nuclear factor kappa-B (NF-κB) signaling, which further up-regulates TRIM47. We showed that protein kinase C epsilon (PKC-ε) and protein kinase D3 (PKD3), known as NF-κB–activating protein kinases, are directly associated with TRIM47 and stabilized in the presence of TRIM47. As an underlying mechanism, we showed TRIM47-dependent lysine 27–linked polyubiquitination of PKC-ε. These results indicate that TRIM47 facilitates breast cancer proliferation and endocrine therapy resistance by forming a ternary complex with PKC-ε and PKD3. TRIM47 and its associated kinases can be a potential diagnostic and therapeutic target for breast cancer refractory to endocrine therapy.

scholarly journals WeFaceNano: a user-friendly pipeline for complete ONT sequence assembly and detection of antibiotic resistance in multi-plasmid bacterial isolates

2021 ◽  
Vol 21 (1) ◽  
Author(s):  
Astrid P. Heikema ◽  
Rick Jansen ◽  
Saskia D. Hiltemann ◽  
John P. Hays ◽  
Andrew P. Stubbs
Keyword(s):  
Antibiotic Resistance ◽  
Resistance Genes ◽  
Dna Sequences ◽  
Sequence Data ◽  
Antibiotic Resistance Genes ◽  
Clinical Samples ◽  
Microbial Resistance ◽  
Sequencing Platform ◽  
Long Read ◽  
User Friendly

Abstract Background Bacterial plasmids often carry antibiotic resistance genes and are a significant factor in the spread of antibiotic resistance. The ability to completely assemble plasmid sequences would facilitate the localization of antibiotic resistance genes, the identification of genes that promote plasmid transmission and the accurate tracking of plasmid mobility. However, the complete assembly of plasmid sequences using the currently most widely used sequencing platform (Illumina-based sequencing) is restricted due to the generation of short sequence lengths. The long-read Oxford Nanopore Technologies (ONT) sequencing platform overcomes this limitation. Still, the assembly of plasmid sequence data remains challenging due to software incompatibility with long-reads and the error rate generated using ONT sequencing. Bioinformatics pipelines have been developed for ONT-generated sequencing but require computational skills that frequently are beyond the abilities of scientific researchers. To overcome this challenge, the authors developed ‘WeFaceNano’, a user-friendly Web interFace for rapid assembly and analysis of plasmid DNA sequences generated using the ONT platform. WeFaceNano includes: a read statistics report; two assemblers (Miniasm and Flye); BLAST searching; the detection of antibiotic resistance- and replicon genes and several plasmid visualizations. A user-friendly interface displays the main features of WeFaceNano and gives access to the analysis tools. Results Publicly available ONT sequence data of 21 plasmids were used to validate WeFaceNano, with plasmid assemblages and anti-microbial resistance gene detection being concordant with the published results. Interestingly, the “Flye” assembler with “meta” settings generated the most complete plasmids. Conclusions WeFaceNano is a user-friendly open-source software pipeline suitable for accurate plasmid assembly and the detection of anti-microbial resistance genes in (clinical) samples where multiple plasmids can be present.

scholarly journals R2C2: Improving nanopore read accuracy enables the sequencing of highly-multiplexed full-length single-cell cDNA

2018 ◽  
Author(s):  
Roger Volden ◽  
Theron Palmer ◽  
Ashley Byrne ◽  
Charles Cole ◽  
Robert J Schmitz ◽  
...  
Keyword(s):  
Quantitative Analysis ◽  
Single Cell ◽  
Cancer Biology ◽  
Full Length ◽  
Short Read ◽  
Transcript Isoforms ◽  
Short Read Sequencing ◽  
Sequencing Method ◽  
Long Read ◽  
Rna Transcript

AbstractHigh-throughput short-read sequencing has revolutionized how transcriptomes are quantified and annotated. However, while Illumina short-read sequencers can be used to analyze entire transcriptomes down to the level of individual splicing events with great accuracy, they fall short of analyzing how these individual events are combined into complete RNA transcript isoforms. Because of this shortfall, long-read sequencing is required to complement short-read sequencing to analyze transcriptomes on the level of full-length RNA transcript isoforms. However, there are issues with both Pacific Biosciences (PacBio) and Oxford Nanopore Technologies (ONT) long-read sequencing technologies that prevent their widespread adoption. Briefly, PacBio sequencers produce low numbers of reads with high accuracy, while ONT sequencers produce higher numbers of reads with lower accuracy. Here we introduce and validate a new long-read ONT based sequencing method. At the same cost, our Rolling Circle Amplification to Concatemeric Consensus (R2C2) method generates more accurate reads of full-length RNA transcript isoforms than any other available long-read sequencing method. These reads can then be used to generate isoform-level transcriptomes for both genome annotation and differential expression analysis in bulk or single cell samples.Significance StatementSubtle changes in RNA transcript isoform expression can have dramatic effects on cellular behaviors in both health and disease. As such, comprehensive and quantitative analysis of isoform-level transcriptomes would open an entirely new window into cellular diversity in fields ranging from developmental to cancer biology. The R2C2 method we are presenting here is the first method with sufficient throughput and accuracy to make the comprehensive and quantitative analysis of RNA transcript isoforms in bulk and single cell samples economically feasible.

scholarly journals IsoSeq transcriptome assembly of C3 panicoid grasses provides tools to study evolutionary change in the Panicoideae

2019 ◽  
Author(s):  
Daniel S. Carvalho ◽  
James C. Schnable
Keyword(s):  
Wild Species ◽  
Sequence Data ◽  
Transcriptome Assembly ◽  
Crop Improvement ◽  
Grass Species ◽  
Crop Species ◽  
Long Read ◽  
Syntenic Gene ◽  
Panicoid Grass ◽  
Reference Genomes

AbstractThe number of plant species with genomic and transcriptomic data has been increasing rapidly. The grasses – Poaceae – have been well represented among species with published reference genomes. However, as a result the genomes of wild grasses are less frequently targeted by sequencing efforts. Sequence data from wild relatives of crop species in the grasses can aid the study of domestication, gene discovery for breeding and crop improvement, and improve our understanding of the evolution of C4 photosynthesis. Here we used long read sequencing technology to characterize the transcriptomes of three C3 panicoid grass species: Dichanthelium oligosanthes, Chasmanthium laxum, and Hymenachne amplexicaulis. Based on alignments to the sorghum genome we estimate that assembled consensus transcripts from each species capture between 54.2 and 65.7% of the conserved syntenic gene space in grasses. Genes co-opted into C4 were also well represented in this dataset, despite concerns that, because these genes might play roles unrelated to photosynthesis in the target species, they would be expressed at low levels and missed by transcript-based sequencing. A combined analysis using syntenic orthologous genes from grasses with published reference genomes and consensus long read sequences from these wild species was consistent with previously published phylogenies. It is hoped that this data, targeting under represented classes of species within the PACMAD grasses – wild species and species utilizing C3 photosynthesis – will aid in futurue studies of domestication and C4 evolution by decreasing the evolutionary distance between C4 and C3 species within this clade, enabling more accurate comparisons associated with evolution of the C4 pathway.

Integrated Quantitative Analysis of the Phosphoproteome and Transcriptome in Tamoxifen-resistant Breast Cancer*

2020 ◽  
Author(s):  
Lungwani Muungo
Keyword(s):  
Breast Cancer ◽  
High Rate ◽  
System Level ◽  
Clinical Samples ◽  
Transcriptome Data ◽  
Data Set ◽  
Reporter Assays ◽  
Treated Breast ◽  
Mcf 7 ◽  
Resistant Cells

Quantitative phosphoproteome and transcriptome analysisof ligand-stimulated MCF-7 human breast cancer cells wasperformed to understand the mechanisms of tamoxifen resistanceat a system level. Phosphoproteome data revealed thatWT cells were more enriched with phospho-proteins thantamoxifen-resistant cells after stimulation with ligands.Surprisingly, decreased phosphorylation after ligand perturbationwas more common than increased phosphorylation.In particular, 17?-estradiol induced down-regulation inWT cells at a very high rate. 17?-Estradiol and the ErbBligand heregulin induced almost equal numbers of up-regulatedphospho-proteins in WT cells. Pathway and motifactivity analyses using transcriptome data additionallysuggested that deregulated activation of GSK3? (glycogensynthasekinase 3?) and MAPK1/3 signaling might be associatedwith altered activation of cAMP-responsive elementbindingprotein and AP-1 transcription factors intamoxifen-resistant cells, and this hypothesis was validatedby reporter assays. An examination of clinical samples revealedthat inhibitory phosphorylation of GSK3? at serine 9was significantly lower in tamoxifen-treated breast cancerpatients that eventually had relapses, implying that activationof GSK3? may be associated with the tamoxifen-resistantphenotype. Thus, the combined phosphoproteomeand transcriptome data set analyses revealed distinct signal

Prognostic Role of Hedgehog-GLI1 Signaling Pathway in Aggressive and Metastatic Breast Cancers

2020 ◽  
Vol 21 (1) ◽  
pp. 33-43 ◽  
Author(s):  
Prasuja Rokkam ◽  
Shailender Gugalavath ◽  
Deepak Kakara Gift Kumar ◽  
Rahul Kumar Vempati ◽  
Rama Rao Malla
Keyword(s):  
Breast Cancer ◽  
Signaling Pathway ◽  
Neural Development ◽  
Splice Variants ◽  
Metastatic Breast ◽  
Basic Function ◽  
Breast Cancers ◽  
Breast Cancer Patients ◽  
Aberrant Expression ◽  
Cellular Processes

Glioma-associated oncogene homolog 1 (GLI1) is reported as an amplified gene in human glioblastoma cells. It is a krupple like transcription factor, belonging to the zinc finger family. The basic function of GLI1 is normal neural development at various stages of human. The GLI1 gene was first mapped on the chromosome sub-bands 12q13.3-14.1. Further, single nucleotide polymorphism is mostly observed in translating a region of 5’ and 3’- UTR of GLI1 gene in addition to two post-transcriptional splice variants, GLIΔN and tGLI. Additionally, it also regulates a plethora of gene which mediates crucial cellular processes like proliferation, differentiation, oncogenesis, EMT, and metastasis. It also regulates tumor tolerance, chemoresistance, and radioresistance. Aberrant expression of GLI1 predicts the poor survival of breast cancer patients. GLI1 is an essential mediator of the SHH signaling pathway regulating self-renewal of stem cells, angiogenesis, and expression of FOXS1, CYR61. GLI1 mediated HH pathway can induce apoptosis. Hence, GLI1 can be a future diagnostic, prognostic marker, and as well as a potent target of therapeutics in breast cancer.

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