Genomics

Comparative genomic analysis of two brucellaphages of distant origins.

Publication Date: 2012 Jan 21 PMID: 22300630
Authors: Flores, V. - Lopez-Merino, A. - Mendoza-Hernandez, G. - Guarneros, G.
Journal: Genomics

Here, we present the first complete genome sequence of brucellaphage Tbilisi (Tb) and compared it with that of Pr, a broad host-range brucellaphage recently isolated in Mexico. The genomes consist of 41,148bp (Tb) and 38,253bp (Pr), they differ mainly in the region encoding structural proteins, in which the genome of Tb shows two major insertions. Both genomes share 99.87% nucleotide identity, a high percentage of identity among phages isolated at so globally distant locations and temporally different occasions. Sequence analysis revealed 57 conserved ORFs, three transcriptional terminators and four putative transcriptional promoters. The co-occurrence of an ORF encoding a putative DnaA-like protein and a putative oriC-like origin of replication was found in both brucellaphages genomes, a feature not described in any other phage genome. These elements suggest that DNA replication in brucellaphages differs from other phages, and might resemble that of bacterial chromosomes.

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Multiplex PCR-based Alu insertion polymorphisms genotyping for identifying individuals of Japanese ethnicity.

Publication Date: 2012 Jan 26 PMID: 22293435
Authors: Asari, M. - Omura, T. - Oka, K. - Maseda, C. - Tasaki, Y. - Shiono, H. - Matsubara, K. - Matsuda, M. - Shimizu, K.
Journal: Genomics

Discrimination of Alu insertions is a useful tool for geographic ancestry analysis, and is usually performed by Alu element amplification and agarose gel electrophoresis. Here, we have developed a new fluorescence-based method for multiple Alu genotyping in forensic identification. Allele frequencies were determined in 70 Japanese individuals, and we selected 30 polymorphic Alu insertions. Three primers were designed for each Alu locus to discriminate alleles using the 3-6bp differences in amplicon sizes. Furthermore, we classified the amplification primers for the 30 loci into three different sets, and PCR using each set of primers provided 10 loci fragments ranging from 50 to 137bp. Based on population data, the probability of incorrectly assigning a match was 3.7x10(-13). Three independent amplifications and subsequent capillary electrophoresis enabled the sensitive genotyping of small amounts of DNA, indicating that this method is suitable for identifying individuals of Japanese ethnicity.

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Whole-exome sequencing in a single proband reveals a mutation in the CHST8 gene in autosomal recessive peeling skin syndrome.

Publication Date: 2012 Jan 25 PMID: 22289416
Authors: Cabral, R. M. - Kurban, M. - Wajid, M. - Shimomura, Y. - Petukhova, L. - Christiano, A. M.
Journal: Genomics

Generalized peeling skin syndrome (PSS) is an autosomal recessive genodermatosis characterized by lifelong, continuous shedding of the upper epidermis. Using whole-genome homozygozity mapping and whole-exome sequencing, we identified a novel homozygous missense mutation (c.229C>T, R77W) within the CHST8 gene, in a large consanguineous family with non-inflammatory PSS type A. CHST8 encodes a Golgi transmembrane N-acetylgalactosamine-4-O-sulfotransferase (GalNAc4-ST1), which we show by immunofluorescence staining to be expressed throughout normal epidermis. A colorimetric assay for total sulfated glycosaminoglycan (GAG) quantification, comparing human keratinocytes (CCD1106 KERTr) expressing wild type and mutant recombinant GalNAc4-ST1, revealed decreased levels of total sulfated GAGs in cells expressing mutant GalNAc4-ST1, suggesting loss of function. Western blotting revealed lower expression levels of mutant recombinant GalNAc4-ST1 compared to wild type, suggesting that accelerated degradation may result in loss of function, leading to PSS type A. This is the first report describing a mutation as the cause of PSS type A.

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Development of TaqMan allelic discrimination based genotyping of large DNA deletions.

Publication Date: 2012 Jan 17 PMID: 22281206
Authors: Fedick, A. - Su, J. - Treff, N. R.
Journal: Genomics

The high prevalence of genetic diseases resulting from gross deletions has highlighted a need for a quick, simple, and reliable method of genotyping these mutations. Here, we developed a novel strategy for applying TaqMan allelic discrimination to accurately genotype 3 different large deletions in a high-throughput manner. Allelic discrimination has previously been used to genotype frame shift and point mutations, and small insertions or deletions six base pairs in length, but not large deletions. The assays designed here recognize a 2502 base pair deletion in the Nebulin (NEB) gene that results in Nemaline Myopathy, a 308,769 base pair deletion in the Gap Junction Protein, beta 6 (GJB6) gene that causes Hearing Loss, and a 6433 base pair deletion in the Mucolipin 1 (MCOLN1) gene responsible for causing Mucolipidosis IV Disease. This methodology may also be successfully applied to high throughput genotyping of other large deletions.

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Characterization of DNA methylation and its association with other biological systems in lymphoblastoid cell lines.

Publication Date: 2012 Jan 15 PMID: 22269447
Authors: Zhang, Z. - Liu, J. - Kaur, M. - Krantz, I. D.
Journal: Genomics

Lymphoblastoid cell line (LCL) is a common tool to study genetic disorders. However, it has not been fully characterized to what degree LCLs preserve the in vivo status of non-genetic biological systems, such as DNA methylation and gene transcription. We previously reported that DNA methylation in LCLs is highly variable in a data set of ~27,000 CpG dinucleotide sites around transcription start site (TSS) and 63 human subjects including healthy controls and probands of genetic disorders. Disease-causing mutations are linked to differential methylation at some CpG sites, but account for a small proportion of the total variance. In this study, we repeated the experiments to ensure that the high variance is not due to technical error and scrutinized the characteristics of DNA methylation and its association with other biological systems. Using sequence information and ChIP-seq data, we conclude that local CpG density and histone modifications not only correlate to baseline methylation level, but also affect the direction of methylation change in LCLs. Integrative analysis of gene transcription and DNA methylation data of the same subjects shows that medium or high methylation around TSS blocks the transcription while low methylation is a necessary, but not sufficient condition of downstream gene transcription. We utilized epigenetic information around TSS to predict active gene transcription via logistic regression models. The multivariate model using DNA methylation, eight histone modifications, and two regulatory protein complexes (CTCF and cohesin) as predictors has better performance (accuracy=95.1%) than any univariate models of single predictors. Linear regression analysis further shows that the transcriptional levels predicted by epigenetic markers have significant correlation to microarray measurements (p=2.2e-10). This study provides new insights into the epigenetic systems of LCLs and suggests that more specifically designed experiments are needed to improve our understanding on this topic.

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Developmental and insecticide-resistant insights from the de novo assembled transcriptome of the diamondback moth, Plutella xylostella.

Publication Date: 2012 Jan 5 PMID: 22240003
Authors: He, W. - You, M. - Vasseur, L. - Yang, G. - Xie, M. - Cui, K. - Bai, J. - Liu, C. - Li, X. - Xu, X. - Huang, S.
Journal: Genomics

We present here the de novo assembly and annotation of the transcriptome of Plutella xylostella (diamondback moth (DBM)), a widespread destructive pest of cruciferous plants, using short reads generated by Illumina sequencing from different developmental stages and insecticide-resistant strains. A total of 171,262 non-redundant sequences, denoted as unigenes, were obtained. They represented approximately 100-fold of all DBM mRNA and EST sequences in GenBank thus far. We identified 38,255 unigenes highly similar to the known functional protein-coding genes, most of which were annotated using gene ontology (GO) and orthologous groups of proteins (COG). Global profiling of differentially expressed unigenes revealed enriched GOs and biological pathways that were related to specific developmental stages and insecticide resistance. We also evaluated the resistance-related single nucleotide polymorphism (SNP) using this high-throughput genotyping method. The newly developed transcriptome will facilitate researches on the DBM developmental biology and insecticide resistance evolution, and ultimately provide better pest management systems.

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