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author:("Akan, peline")
1.  TagGD: Fast and Accurate Software for DNA Tag Generation and Demultiplexing 
PLoS ONE  2013;8(3):e57521.
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).
doi:10.1371/journal.pone.0057521
PMCID: PMC3587622  PMID: 23469199
2.  Comprehensive analysis of the genome transcriptome and proteome landscapes of three tumor cell lines 
Genome Medicine  2012;4(11):86.
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.
doi:10.1186/gm387
PMCID: PMC3580420  PMID: 23158748
3.  Comparison of total and cytoplasmic mRNA reveals global regulation by nuclear retention and miRNAs 
BMC Genomics  2012;13:574.
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.
doi:10.1186/1471-2164-13-574
PMCID: PMC3495644  PMID: 23110385
Differential detection; Gene expression; Nuclear retention; miRNA regulation; RNA-Seq
4.  Exploration of signals of positive selection derived from genotype-based human genome scans using re-sequencing data 
Human Genetics  2011;131(5):665-674.
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.
Electronic supplementary material
The online version of this article (doi:10.1007/s00439-011-1111-9) contains supplementary material, which is available to authorized users.
doi:10.1007/s00439-011-1111-9
PMCID: PMC3325425  PMID: 22057783
5.  Integrated Genetic and Epigenetic Analysis Identifies Haplotype-Specific Methylation in the FTO Type 2 Diabetes and Obesity Susceptibility Locus 
PLoS ONE  2010;5(11):e14040.
Recent multi-dimensional approaches to the study of complex disease have revealed powerful insights into how genetic and epigenetic factors may underlie their aetiopathogenesis. We examined genotype-epigenotype interactions in the context of Type 2 Diabetes (T2D), focussing on known regions of genomic susceptibility. We assayed DNA methylation in 60 females, stratified according to disease susceptibility haplotype using previously identified association loci. CpG methylation was assessed using methylated DNA immunoprecipitation on a targeted array (MeDIP-chip) and absolute methylation values were estimated using a Bayesian algorithm (BATMAN). Absolute methylation levels were quantified across LD blocks, and we identified increased DNA methylation on the FTO obesity susceptibility haplotype, tagged by the rs8050136 risk allele A (p = 9.40×10−4, permutation p = 1.0×10−3). Further analysis across the 46 kb LD block using sliding windows localised the most significant difference to be within a 7.7 kb region (p = 1.13×10−7). Sequence level analysis, followed by pyrosequencing validation, revealed that the methylation difference was driven by the co-ordinated phase of CpG-creating SNPs across the risk haplotype. This 7.7 kb region of haplotype-specific methylation (HSM), encapsulates a Highly Conserved Non-Coding Element (HCNE) that has previously been validated as a long-range enhancer, supported by the histone H3K4me1 enhancer signature. This study demonstrates that integration of Genome-Wide Association (GWA) SNP and epigenomic DNA methylation data can identify potential novel genotype-epigenotype interactions within disease-associated loci, thus providing a novel route to aid unravelling common complex diseases.
doi:10.1371/journal.pone.0014040
PMCID: PMC2987816  PMID: 21124985
7.  Linkage Disequilibrium Mapping of the Replicated Type 2 Diabetes Linkage Signal on Chromosome 1q 
Diabetes  2009;58(7):1704-1709.
OBJECTIVE
Linkage of the chromosome 1q21–25 region to type 2 diabetes has been demonstrated in multiple ethnic groups. We performed common variant fine-mapping across a 23-Mb interval in a multiethnic sample to search for variants responsible for this linkage signal.
RESEARCH DESIGN AND METHODS
In all, 5,290 single nucleotide polymorphisms (SNPs) were successfully genotyped in 3,179 type 2 diabetes case and control subjects from eight populations with evidence of 1q linkage. Samples were ascertained using strategies designed to enhance power to detect variants causal for 1q linkage. After imputation, we estimate ∼80% coverage of common variation across the region (r 2 > 0.8, Europeans). Association signals of interest were evaluated through in silico replication and de novo genotyping in ∼8,500 case subjects and 12,400 control subjects.
RESULTS
Association mapping of the 23-Mb region identified two strong signals, both of which were restricted to the subset of European-descent samples. The first mapped to the NOS1AP (CAPON) gene region (lead SNP: rs7538490, odds ratio 1.38 [95% CI 1.21–1.57], P = 1.4 × 10−6, in 999 case subjects and 1,190 control subjects); the second mapped within an extensive region of linkage disequilibrium that includes the ASH1L and PKLR genes (lead SNP: rs11264371, odds ratio 1.48 [1.18–1.76], P = 1.0 × 10−5, under a dominant model). However, there was no evidence for association at either signal on replication, and, across all data (>24,000 subjects), there was no indication that these variants were causally related to type 2 diabetes status.
CONCLUSIONS
Detailed fine-mapping of the 23-Mb region of replicated linkage has failed to identify common variant signals contributing to the observed signal. Future studies should focus on identification of causal alleles of lower frequency and higher penetrance.
doi:10.2337/db09-0081
PMCID: PMC2699860  PMID: 19389826
8.  Linkage disequilibrium mapping of the replicated type 2 diabetes linkage signal on chromosome 1q 
Diabetes  2009;58(7):1704-1709.
Objective
Linkage of the chromosome 1q21-25 region to type 2 diabetes has been demonstrated in multiple ethnic groups. We performed common variant fine-mapping across a 23Mb interval in a multiethnic sample to search for variants responsible for this linkage signal.
Research Design and Methods
In all, 5,290 SNPs were successfully genotyped in 3,179 T2D cases and controls from eight populations with evidence of 1q linkage. Samples were ascertained using strategies designed to enhance power to detect variants causal for 1q-linkage. Following imputation, we estimate ~80% coverage of common variation across the region (r2>0.8, Europeans). Association signals of interest were evaluated through in silico replication and de novo genotyping in approximately 8,500 cases and 12,400 controls.
Results
Association mapping of the 23Mb region identified two strong signals, both restricted to the subset of European-descent samples. The first mapped to the NOS1AP (CAPON) gene region (lead SNP: rs7538490, OR 1.38 (95% CI, 1.21-1.57), p=1.4×10-6 in 999 cases and 1,190 controls): the second within an extensive region of linkage disequilibrium that includes the ASH1L and PKLR genes (lead SNP: rs11264371, OR 1.48 [1.18-1.76], p=1.0×10-5, under a dominant model). However, there was no evidence for association at either signal on replication, and, across all data (>24,000 subjects), no indication that these variants were causally-related to T2D status.
Conclusion
Detailed fine-mapping of the 23Mb region of replicated linkage has failed to identify common variant signals contributing to the observed signal. Future studies should focus on identification of causal alleles of lower frequency and higher penetrance.
doi:10.2337/db09-0081
PMCID: PMC2699860  PMID: 19389826
chromosome 1q; linkage; association
9.  DNA sequence and structural properties as predictors of human and mouse promoters 
Gene  2007;410(1):165-176.
Promoters play a central role in gene regulation, yet our power to discriminate them from non-promoter sequences in higher eukaryotes is mainly restricted to those associated with CpG islands. Here, we examined in silico the promoters of 30,954 human and 18,083 mouse transcripts in the DBTSS database, to assess the impact of particular sequence and structural features (propeller twist, bendability and nucleosome positioning preference) on promoter classification and prediction. Our analysis showed that a stricter-than-traditional definition of CpG islands captures low and high CpG count promoter classes more accurately than the traditional one. We observed that both human and mouse promoter sequences are flexible with the exception of the TATA box and TSS, which are rigid regions irrespective of association with a CpG island. Therefore varying levels of structural flexibility in promoters may affect their accessibility to proteins, and hence their specificity. For all features investigated, averaged values across core promoters discriminated CpG island associated promoters from background, whereas the same did not hold for promoters without a CpG island. However, local changes around - 34 to - 23 (expected position of TATA box) and the TSS were informative in discriminating promoters (both classes) from non-promoter sequences. Additionally, we investigated ATG deserts and observed that they occur in all promoter sets except those with a TATA-box and without a CpG island in human. Interestingly, all mouse promoter sets showed ATG codon depletion irrespective of the presence of a TATA-box, possibly reflecting a weaker contribution to TSS specificity in mouse.
doi:10.1016/j.gene.2007.12.011
PMCID: PMC2672154  PMID: 18234453
Promoter; Human; Mouse; Genome-wide computational analysis; CpG island; TATA-box; DNA bendability; Propeller twist; Nucleosome positioning preference; ATG desert
10.  A Histone Map of Human Chromosome 20q13.12 
PLoS ONE  2009;4(2):e4479.
Background
We present a systematic search for regulatory elements in a 3.5 Mb region on human chromosome 20q13.12, a region associated with a number of medical conditions such as type II diabetes and obesity.
Methodology/Principal Findings
We profiled six histone modifications alongside RNA polymerase II (PolII) and CTCF in two cell lines, HeLa S3 and NTERA-2 clone D1 (NT2/D1), by chromatin immunoprecipitation using an in-house spotted DNA array, constructed with 1.8 kb overlapping plasmid clones. In both cells, more than 90% of transcription start sites (TSSs) of expressed genes showed enrichments with PolII, di-methylated lysine 4 of histone H3 (H3K4me2), tri-methylated lysine 4 of histone H3 (H3K4me3) or acetylated H3 (H3Ac), whereas mono-methylated lysine 4 of histone H3 (H3K4me1) signals did not correlate with expression. No TSSs were enriched with tri-methylated lysine 27 of histone H3 (H3K27me3) in HeLa S3, while eight TSSs (4 expressed) showed enrichments in NT2/D1. We have also located several CTCF binding sites that are potential insulator elements.
Conclusions/Significance
In summary, we annotated a number of putative regulatory elements in 20q13.12 and went on to verify experimentally a subset of them using dual luciferase reporter assays. Correlating this data to sequence variation can aid identification of disease causing variants.
doi:10.1371/journal.pone.0004479
PMCID: PMC2639704  PMID: 19229332
11.  DNA sequence and structural properties as predictors of human and mouse promoters 
Gene  2008;410(1-2):165-176.
Promoters play a central role in gene regulation, yet our power to discriminate them from non-promoter sequences in higher eukaryotes is mainly restricted to those associated with CpG islands. Here, we examined in silico the promoters of 30,954 human and 18,083 mouse transcripts in the DBTSS database, to assess the impact of particular sequence and structural features (propeller twist, bendability and nucleosome positioning preference) on promoter classification and prediction. Our analysis showed that a stricter-than-traditional definition of CpG islands captures low and high CpG count promoter classes more accurately than the traditional one. We observed that both human and mouse promoter sequences are flexible with the exception of the TATA box and TSS, which are rigid regions irrespective of association with a CpG island. Therefore varying levels of structural flexibility in promoters may affect their accessibility to proteins, and hence their specificity. For all features investigated, averaged values across core promoters discriminated CpG island associated promoters from background, whereas the same did not hold for promoters without a CpG island. However, local changes around − 34 to − 23 (expected position of TATA box) and the TSS were informative in discriminating promoters (both classes) from non-promoter sequences. Additionally, we investigated ATG deserts and observed that they occur in all promoter sets except those with a TATA-box and without a CpG island in human. Interestingly, all mouse promoter sets showed ATG codon depletion irrespective of the presence of a TATA-box, possibly reflecting a weaker contribution to TSS specificity in mouse.
doi:10.1016/j.gene.2007.12.011
PMCID: PMC2672154  PMID: 18234453
TBP, TATA-box binding protein; TSS, transcription start site; CGI, CpG island; WCGI, associated with CpG island; WOCGI, not associated with CpG island; DBTSS, Database of Transcription Start Sites; R, purine; Y, pyrimidine; DPE, downstream promoter element; BRE, TFIIB recognition element; LCG, low CpG count; HCG, high CpG count.; Promoter; Human; Mouse; Genome-wide computational analysis; CpG island; TATA-box; DNA bendability; Propeller twist; Nucleosome positioning preference; ATG desert

Results 1-11 (11)