Hui Jiang, Wing Hung Wong.
Bioinformatics, Advance Access | doi:10.1093/bioinformatics/btp113 | PMID:19244387
The development of RNA sequencing (RNA-Seq) makes it possible for us to measure transcription at an unprecedented precision and throughput. However challenges remain in understanding the source and distribution of the reads, modeling the transcript abundance and developing efficient computational methods. In this paper, we develop a method to deal with the isoform expression estimation problem. The count of reads falling into a locus on the genome annotated with multiple isoforms is modeled as a Poisson variable. The expression of each individual isoform is estimated by solving a convex optimization problem and statistical inferences about the parameters are obtained from the posterior distribution by importance sampling. Our results show that isoform expression inference in RNA-Seq is possible by employing appropriate statistical methods.
Yann Astier, Oktay Uzun, Francesco Stellacci.
Small, Early View | doi:10.1002/smll.200801779 | PMID:19242940
Single-monolayer-protected gold nanoparticles can be captured in the α-hemolysin nanopore (see image). Single-nanopore ion conductance studies of the nanoparticle/nanopore complex are described. The effect of the nanoparticle size, charge, and surface coating on ssDNA threading speed through the nanopore/nanoparticle complex is discussed.
QSRA – a quality-value guided de novo short read assembler.
Douglas W Bryant Jr, Weng-Keen Wong, Todd C Mockler.
BMC Bioinformatics 10, 69 (2009) | doi:10.1186/1471-2105-10-69 | PMID:
Background
New rapid high-throughput sequencing technologies have sparked the creation of a new class of assembler. Since all high-throughput sequencing platforms incorporate errors in their output, short-read assemblers must be designed to account for this error while utilizing all available data.
Results
We have designed and implemented an assembler, Quality-value guided Short Read Assembler, created to take advantage of quality-value scores as a further method of dealing with error. Compared to previous published algorithms, our assembler shows significant improvements not only in speed but also in output quality.
Conclusion
QSRA generally produced the highest genomic coverage, while being faster than VCAKE. QSRA is extremely competitive in its longest contig and N50/N80 contig lengths, producing results of similar quality to those of EDENA and VELVET. QSRA provides a step closer to the goal of de novo assembly of complex genomes, improving upon the original VCAKE algorithm by not only drastically reducing runtimes but also increasing the viability of the assembly algorithm through further error handling capabilities.
Alterations in Genes of the EGFR Signaling Pathway and Their Relationship to EGFR Tyrosine Kinase Inhibitor Sensitivity in Lung Cancer Cell Lines.
Jeet Gandhi, Jianling Zhang, Yang Xie, Junichi Soh, Hisayuki Shigematsu, Wei Zhang, Hiromasa Yamamoto, Michael Peyton, Luc Girard, William W. Lockwood, Wan L. Lam, Marileila Varella-Garcia, John D. Minna, Adi F. Gazdar.
PLoS ONE 4 e4576 (2009) | doi:10.1371/journal.pone.0004576 | PMID:19238210
Background
Deregulation of EGFR signaling is common in non-small cell lung cancers (NSCLC) and this finding led to the development of tyrosine kinase inhibitors (TKIs) that are highly effective in a subset of NSCLC. Mutations of EGFR (mEGFR) and copy number gains (CNGs) of EGFR (gEGFR) and HER2 (gHER2) have been reported to predict for TKI response. Mutations in KRAS (mKRAS) are associated with primary resistance to TKIs.
Methodology/Principal Findings
We investigated the relationship between mutations, CNGs and response to TKIs in a large panel of NSCLC cell lines. Genes studied were EGFR, HER2, HER3, HER4, KRAS, BRAF and PIK3CA. Mutations were detected by sequencing, while CNGs were determined by quantitative PCR (qPCR), fluorescence in situ hybridization (FISH) and array comparative genomic hybridization (aCGH). IC50 values for the TKIs gefitinib (Iressa) and erlotinib (Tarceva) were determined by MTS assay. For any of the seven genes tested, mutations (39/77, 50.6%), copy number gains (50/77, 64.9%) or either (65/77, 84.4%) were frequent in NSCLC lines. Mutations of EGFR (13%) and KRAS (24.7%) were frequent, while they were less frequent for the other genes. The three techniques for determining CNG were well correlated, and qPCR data were used for further analyses. CNGs were relatively frequent for EGFR and KRAS in adenocarcinomas. While mutations were largely mutually exclusive, CNGs were not. EGFR and KRAS mutant lines frequently demonstrated mutant allele specific imbalance i.e. the mutant form was usually in great excess compared to the wild type form. On a molar basis, sensitivity to gefitinib and erlotinib were highly correlated. Multivariate analyses led to the following results:
Conclusions/Significance
- mEGFR and gEGFR and gHER2 were independent factors related to gefitinib sensitivity, in descending order of importance.
- mKRAS was associated with increased in vitro resistance to gefitinib.
Our in vitro studies confirm and extend clinical observations and demonstrate the relative importance of both EGFR mutations and CNGs and HER2 CNGs in the sensitivity to TKIs.
Katsuya Tsuchihara, Yutaka Suzuki, Hiroyuki Wakaguri, Takuma Irie, Kousuke Tanimoto, Shin-ichi Hashimoto, Kouji Matsushima, Junko Mizushima-Sugano, Riu Yamashita, Kenta Nakai, David Bentley, Hiroyasu Esumi, Sumio Sugano.
Nucleic Acids Research, Advance Access | doi:10.1093/nar/gkp066 | PMID:19237398
Combining our full-length cDNA method and the massively parallel sequencing technology, we developed a simple method to collect precise positional information of transcriptional start sites (TSSs) together with digital information of the gene-expression levels in a high throughput manner. We applied this method to observe gene-expression changes in a colon cancer cell line cultured in normoxic and hypoxic conditions. We generated more than 100 million 36-base TSS-tag sequences and revealed comprehensive features of hypoxia responsive alterations in the transcriptional landscape of the human genome. The features include presence of inducible ‘hot regions’ in 54 genomic regions, 220 novel hypoxia inducible promoters that may drive non-protein-coding transcripts, 191 hypoxia responsive alternative promoters and detailed views of 120 novel as well as known hypoxia responsive genes. We further analyzed hypoxic response of different cells using additional 60 million TSS-tags and found that the degree of the gene-expression changes were different among cell lines, possibly reflecting cellular robustness against hypoxia. The novel dynamic figure of the human gene transcriptome will deepen our understanding of the transcriptional program of the human genome as well as bringing new insights into the biology of cancer cells in hypoxia.
Novel sequencing strategy for repetitive DNA in a Drosophila BAC clone reveals that the centromeric region of the Y chromosome evolved from a telomere.
María Méndez-Lago, Jadwiga Wild, Siobhan L. Whitehead, Alan Tracey, Beatriz de Pablos, Jane Rogers, Waclaw Szybalski, Alfredo Villasante.
Nucleic Acids Research, Advance Access | doi:10.1093/nar/gkp085 | PMID:19237394
The centromeric and telomeric heterochromatin of eukaryotic chromosomes is mainly composed of middle-repetitive elements, such as transposable elements and tandemly repeated DNA sequences. Because of this repetitive nature, Whole Genome Shotgun Projects have failed in sequencing these regions. We describe a novel kind of transposon-based approach for sequencing highly repetitive DNA sequences in BAC clones. The key to this strategy relies on physical mapping the precise position of the transposon insertion, which enables the correct assembly of the repeated DNA. We have applied this strategy to a clone from the centromeric region of the Y chromosome of Drosophila melanogaster. The analysis of the complete sequence of this clone has allowed us to prove that this centromeric region evolved from a telomere, possibly after a pericentric inversion of an ancestral telocentric chromosome. Our results confirm that the use of transposon-mediated sequencing, including positional mapping information, improves current finishing strategies. The strategy we describe could be a universal approach to resolving the heterochromatic regions of eukaryotic genomes.
