| 1. |
- Akan, Pelin, et al.
(författare)
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Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines
- 2012
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Ingår i: Genome Medicine. - : Springer Science and Business Media LLC. - 1756-994X. ; 4, s. 86-
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Tidskriftsartikel (refereegranskat)abstract
- We here present a comparative genome, transcriptome and functional network analysis of three human cancer cell lines (A431, U251MG and U2OS), and investigate their relation to protein expression. Gene copy numbers significantly influenced corresponding transcript levels; their effect on protein levels was less pronounced. We focused on genes with altered mRNA and/or protein levels to identify those active in tumor maintenance. We provide comprehensive information for the three genomes and demonstrate the advantage of integrative analysis for identifying tumor-related genes amidst numerous background mutations by relating genomic variation to expression/protein abundance data and use gene networks to reveal implicated pathways.
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| 2. |
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| 3. |
- Costea, Paul Igor, et al.
(författare)
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TagGD : Fast and Accurate Software for DNA Tag Generation and Demultiplexing
- 2013
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Ingår i: PLOS ONE. - : Public Library of Science (PLoS). - 1932-6203. ; 8:3, s. e57521-
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Tidskriftsartikel (refereegranskat)abstract
- Multiplexing is of vital importance for utilizing the full potential of next generation sequencing technologies. We here report TagGD (DNA-based Tag Generator and Demultiplexor), a fully-customisable, fast and accurate software package that can generate thousands of barcodes satisfying user-defined constraints and can guarantee full demultiplexing accuracy. The barcodes are designed to minimise their interference with the experiment. Insertion, deletion and substitution events are considered when designing and demultiplexing barcodes. 20,000 barcodes of length 18 were designed in 5 minutes and 2 million barcoded Illumina HiSeq-like reads generated with an error rate of 2% were demultiplexed with full accuracy in 5 minutes. We believe that our software meets a central demand in the current high-throughput biology and can be utilised in any field with ample sample abundance. The software is available on GitHub (https://github.com/pelinakan/UBD.git).
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| 4. |
- Hu, M., et al.
(författare)
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Exploration of signals of positive selection derived from genotype-based human genome scans using re-sequencing data
- 2012
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Ingår i: Human Genetics. - : Springer Science and Business Media LLC. - 0340-6717 .- 1432-1203. ; 131:5, s. 665-674
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Tidskriftsartikel (refereegranskat)abstract
- We have investigated whether regions of the genome showing signs of positive selection in scans based on haplotype structure also show evidence of positive selection when sequence-based tests are applied, whether the target of selection can be localized more precisely, and whether such extra evidence can lead to increased biological insights. We used two tools: simulations under neutrality or selection, and experimental investigation of two regions identified by the HapMap2 project as putatively selected in human populations. Simulations suggested that neutral and selected regions should be readily distinguished and that it should be possible to localize the selected variant to within 40 kb at least half of the time. Re-sequencing of two ∼300 kb regions (chr4:158Mb and chr10:22Mb) lacking known targets of selection in HapMap CHB individuals provided strong evidence for positive selection within each and suggested the micro-RNA gene hsa-miR-548c as the best candidate target in one region, and changes in regulation of the sperm protein gene SPAG6 in the other.
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| 5. |
- Sánchez, J. L. A., et al.
(författare)
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Multiplex PCB-based electrochemical detection of cancer biomarkers using MLPA-barcode approach
- 2016
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Ingår i: Biosensors & bioelectronics. - : Elsevier. - 0956-5663 .- 1873-4235. ; 82, s. 224-232
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Tidskriftsartikel (refereegranskat)abstract
- Asymmetric multiplex ligation-dependent probe amplification (MLPA) was developed for the amplification of seven breast cancer related mRNA markers and the MLPA products were electrochemically detected via hybridization. Seven breast cancer genetic markers were amplified by means of the MLPA reaction, which allows for multiplex amplification of multiple targets with a single primer pair. Novel synthetic MLPA probes were designed to include a unique barcode sequence in each amplified gene. Capture probes complementary to each of the barcode sequences were immobilized on each electrode of a low-cost electrode microarray manufactured on standard printed circuit board (PCB) substrates. The functionalised electrodes were exposed to the single-stranded MLPA products and following hybridization, a horseradish peroxidase (HRP)-labelled DNA secondary probe complementary to the amplified strand completed the genocomplex, which was electrochemically detected following substrate addition. The electrode arrays fabricated using PCB technology exhibited an excellent electrochemical performance, equivalent to planar photolithographically-fabricated gold electrodes, but at a vastly reduced cost (>50 times lower per array). The optimised system was demonstrated to be highly specific with negligible cross-reactivity allowing the simultaneous detection of the seven mRNA markers, with limits of detections as low as 25 pM. This approach provides a novel strategy for the genetic profiling of tumour cells via integrated "amplification-to-detection".
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| 6. |
- Shirley, B. C., et al.
(författare)
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Interpretation, Stratification and Evidence for Sequence Variants Affecting mRNA Splicing in Complete Human Genome Sequences
- 2013
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Ingår i: Genomics, Proteomics and Bioinformatics. - : Elsevier BV. - 1672-0229. ; 11:2, s. 77-85
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Tidskriftsartikel (refereegranskat)abstract
- Information theory-based methods have been shown to be sensitive and specific for predicting and quantifying the effects of non-coding mutations in Mendelian diseases. We present the Shannon pipeline software for genome-scale mutation analysis and provide evidence that the software predicts variants affecting mRNA splicing. Individual information contents (in bits) of reference and variant splice sites are compared and significant differences are annotated and prioritized. The software has been implemented for CLC-Bio Genomics platform. Annotation indicates the context of novel mutations as well as common and rare SNPs with splicing effects. Potential natural and cryptic mRNA splicing variants are identified, and null mutations are distinguished from leaky mutations. Mutations and rare SNPs were predicted in genomes of three cancer cell lines (U2OS, U251 and A431), which were supported by expression analyses. After filtering, tractable numbers of potentially deleterious variants are predicted by the software, suitable for further laboratory investigation. In these cell lines, novel functional variants comprised 6-17 inactivating mutations, 1-5 leaky mutations and 6-13 cryptic splicing mutations. Predicted effects were validated by RNA-seq analysis of the three aforementioned cancer cell lines, and expression microarray analysis of SNPs in HapMap cell lines.
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| 7. |
- Stranneheim, Henrik, et al.
(författare)
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Transcript nuclear retention effects quantification of gene expression levels
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Annan publikation (övrigt vetenskapligt/konstnärligt)abstract
- The majority of published differential gene expression studies have used RNA isolated from whole cell extracts (total RNA), overlooking the potential impact of including the nuclear transcriptome in the analyses. It has not been firmly established that the contribution of nuclear RNA is negligible or how the inclusion of it affects quantification of gene expression. Previous studies have estimated that the nuclear transcriptome is five to ten times more complex than the cytoplasmic [1]. Hence, RNA purified solely from the cytoplasm should have fewer unique transcripts, resulting in more sequence counts per transcript and resulting in increased power to detect remaining transcripts. In this study, cytoplasmic and total mRNA have been prepared from three human cell‐lines and sequenced using massive parallel sequencing. The resulting sequence data was analyzed regarding the effect of number of biological replicates, read length and transcripts fractionation on calling differentially detected genes. In addition, the impact of length and secondary structure of mRNAs un‐translated regions (UTRs), and coding sequence length on nucleus to cytoplasm transportation rates of mRNAs was studied. We observe that the number of differentially detected genes was not significantly increased by adding more than three biological replicates or by increasing the sequence read length > 35bp. More differentially detected genes were found in the cytoplasmic RNA compared to the total RNA and a nuclear retention effect was observed for transcripts with long and structured 5’‐ and 3’‐UTR or long protein coding sequences.
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| 8. |
- Werne Solnestam, Beata, et al.
(författare)
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Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs
- 2012
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Ingår i: BMC Genomics. - : Springer Science and Business Media LLC. - 1471-2164. ; 13:1, s. 574-
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Tidskriftsartikel (refereegranskat)abstract
- Background: The majority of published gene-expression studies have used RNA isolated from whole cells, overlooking the potential impact of including nuclear transcriptome in the analyses. In this study, mRNA fractions from the cytoplasm and from whole cells (total RNA) were prepared from three human cell lines and sequenced using massive parallel sequencing. Results: For all three cell lines, of about 15000 detected genes approximately 400 to 1400 genes were detected in different amounts in the cytoplasmic and total RNA fractions. Transcripts detected at higher levels in the total RNA fraction had longer coding sequences and higher number of miRNA target sites. Transcripts detected at higher levels in the cytoplasmic fraction were shorter or contained shorter untranslated regions. Nuclear retention of transcripts and mRNA degradation via miRNA pathway might contribute to this differential detection of genes. The consequence of the differential detection was further investigated by comparison to proteomics data. Interestingly, the expression profiles of cytoplasmic and total RNA correlated equally well with protein abundance levels indicating regulation at a higher level. Conclusions: We conclude that expression levels derived from the total RNA fraction be regarded as an appropriate estimate of the amount of mRNAs present in a given cell population, independent of the coding sequence length or UTRs.
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