Nonoptical massive parallel DNA sequencing of BRCA1 and BRCA2 genes in a diagnostic setting.
AuthorsCosta JL, Sousa S, Justino A, Kay T, Fernandes S, Cirnes L, Schmitt F, Machado JC
JournalHum Mutat
PubMed ID23315985
'The introduction of the benchtop massive parallel sequencers made it possible for the majority of clinical diagnostic laboratories to gain access to this fast evolving technology. In this study, using the Ion Torrent Personal Genome Machine, we present a strategy for the molecular diagnosis of hereditary breast and ovarian cancer ... More
Whole-Genome Profiling of a Novel Mutagenesis Technique Using Proofreading-Deficient DNA Polymerase d.
AuthorsShiwa Y, Fukushima-Tanaka S, Kasahara K, Horiuchi T, Yoshikawa H
JournalInt J Evol Biol
PubMed ID22675654
'A novel mutagenesis technique using error-prone DNA polymerase d (pold), the disparity mutagenesis model of evolution, has been successfully employed to generate novel microorganism strains with desired traits. However, little else is known about the spectra of mutagenic effects caused by disparity mutagenesis. We evaluated and compared the performance of ... More
Amino-acid substitutions at the domain interface affect substrate and allosteric inhibitor binding in a-isopropylmalate synthase from Mycobacterium tuberculosis.
AuthorsHuisman FH, Squire CJ, Parker EJ
JournalBiochem Biophys Res Commun
PubMed ID23500460
'a-Isopropylmalate synthase (a-IPMS) is a multi-domain protein catalysing the condensation of a-ketoisovalerate (a-KIV) and acetyl coenzyme A (AcCoA) to form a-isopropylmalate. This reaction is the first committed step in the leucine biosynthetic pathway in bacteria and plants, and a-IPMS is allosterically regulated by this amino acid. Existing crystal structures of ... More
Amino acid sequence of the ligand-binding domain of the aryl hydrocarbon receptor 1 predicts sensitivity of wild birds to effects of dioxin-like compounds.
'The sensitivity of avian species to the toxic effects of dioxin-like compounds (DLCs) varies up to 1000-fold among species, and this variability has been associated with interspecies differences in aryl hydrocarbon receptor 1 ligand-binding domain (AHR1 LBD) sequence. We previously showed that LD(50) values, based on in ovo exposures to ... More
Ultrasensitive detection of rare mutations using next-generation targeted resequencing.
AuthorsFlaherty P, Natsoulis G, Muralidharan O, Winters M, Buenrostro J, Bell J, Brown S, Holodniy M, Zhang N, Ji HP
JournalNucleic Acids Res
PubMed ID22013163
'With next-generation DNA sequencing technologies, one can interrogate a specific genomic region of interest at very high depth of coverage and identify less prevalent, rare mutations in heterogeneous clinical samples. However, the mutation detection levels are limited by the error rate of the sequencing technology as well as by the ... More
Quality control methods for optimal BCR-ABL1 clinical testing in human whole blood samples.
'Reliable breakpoint cluster region (BCR)--Abelson (ABL) 1 measurement is essential for optimal management of chronic myelogenous leukemia. There is a need to optimize quality control, sensitivity, and reliability of methods used to measure a major molecular response and/or treatment failure. The effects of room temperature storage time, different primers, and ... More
Genomic approaches for determining nucleosome occupancy in yeast.
AuthorsTsui K, Durbic T, Gebbia M, Nislow C
JournalMethods Mol Biol
PubMed ID22183606
'The basic unit of chromatin is double-stranded DNA wrapped around nucleosome core particles, the -classic "beads-on-a-string" described by Kornberg and colleagues. The history of chromatin studies has experienced many peaks, from the earliest studies by Miescher to the biochemical studies of the 1960s and 1970s, the appreciation for the influence ... More
Genome-wide approaches to determining nucleosome occupancy in metazoans using MNase-Seq.
AuthorsCui K, Zhao K
JournalMethods Mol Biol
PubMed ID22183607
'The precise location of nucleosomes in functional regulatory regions in chromatin is critical to the regulation of transcription. The nucleosome structure protects DNA from microccocal nuclease (MNase) digestion and leaves a footprint on DNA that indicates the position of nucleosomes. Short sequence reads (25-36 bp) from ends of mononucleosome-sized DNA ... More
Genetic diversity of subgroup 1 ilarviruses from eastern Australia.
AuthorsSharman M, Thomas JE
JournalArch Virol
PubMed ID23474983
'This is the first report of the genetic diversity within ilarvirus subgroup 1 from eastern Australia. It supports the separation of tobacco streak virus (TSV) strains from parthenium (Parthenium hysterophorus) and crownbeard (Verbescina encelioides) based on serology and host specificity. It has confirmed one previously described strain of TSV as ... More
In vitro phenotyping of recombinant hepatitis B virus containing the polymerase/reverse transcriptase gene from clinical isolates.
AuthorsLiu Y, Kitrinos KM
Journal
PubMed ID23821268
'This chapter describes the procedures for the generation of recombinant hepatitis B virus (HBV) that contains the polymerase/reverse transcriptase (pol/RT) gene from clinical isolates. It also contains procedures for phenotypic testing of the recombinant HBV in HepG2 cells for susceptibility to nucleos(t)ide analogs. Amplification and cloning of the pol/RT gene ... More
Capturing chloroplast variation for molecular ecology studies: a simple next generation sequencing approach applied to a rainforest tree.
AuthorsMcPherson H, van der Merwe M, Delaney SK, Edwards MA, Henry RJ, McIntosh E, Rymer PD, Milner ML, Siow J, Rossetto M
JournalBMC Ecol
PubMed ID23497206
'With high quantity and quality data production and low cost, next generation sequencing has the potential to provide new opportunities for plant phylogeographic studies on single and multiple species. Here we present an approach for in silicio chloroplast DNA assembly and single nucleotide polymorphism detection from short-read shotgun sequencing. The ... More
Quantification of microRNA Expression with Next-Generation Sequencing.
AuthorsEminaga S, Christodoulou DC, Vigneault F, Church GM, Seidman JG
Journal
PubMed ID23821442
'Rapid advancement of next-generation sequencing technologies has made it possible to study expression profiles of microRNAs (miRNAs) comprehensively and efficiently. Multiplexing miRNA libraries by barcoding can significantly reduce sequencing cost per sample without compromising library quality. This unit provides a step-by-step protocol for isolating miRNAs and constructing multiplexed miRNA libraries. ... More
Ectopic expression of AtPAD4 broadens resistance of soybean to soybean cyst and root-knot nematodes.
The gene encoding PAD4 (PHYTOALEXIN-DEFICIENT4) is required in Arabidopsis for expression of several genes involved in the defense response to Pseudomonas syringae pv. maculicola. AtPAD4 (Arabidopsis thaliana PAD4) encodes a lipase-like protein that plays a regulatory role mediating salicylic acid signaling. ... More
Identification of hepatitis C virus transmission using a next-generation sequencing approach.
AuthorsEscobar-Gutiérrez A, Vazquez-Pichardo M, Cruz-Rivera M, Rivera-Osorio P, Carpio-Pedroza JC, Ruíz-Pacheco JA, Ruiz-Tovar K, Vaughan G
JournalJ Clin Microbiol
PubMed ID22301026
Here, we describe a transmission event of hepatitis C virus (HCV) among injection drug users. Next-generation sequencing (NGS) was used to assess the intrahost viral genetic variation. Deep amplicon sequencing of HCV hypervariable region 1 allowed for a detailed analysis of the structure of the viral population. Establishment of the ... More
Genotyping 1000 yeast strains by next-generation sequencing.
AuthorsWilkening S, Tekkedil MM, Lin G, Fritsch ES, Wei W, Gagneur J, Lazinski DW, Camilli A, Steinmetz LM
JournalBMC Genomics
PubMed ID23394869
The throughput of next-generation sequencing machines has increased dramatically over the last few years; yet the cost and time for library preparation have not changed proportionally, thus representing the main bottleneck for sequencing large numbers of samples. Here we present an economical, high-throughput library preparation method for the Illumina platform, ... More
Analysis of post-blood meal flight distances in mosquitoes utilizing zoo animal blood meals.
We assessed the post-blood meal flight distance of four mosquito species in a unique environment using blood meal analysis. Mosquitoes were trapped at the Rio Grande Zoo in Albuquerque, NM, and the blood source of blood-engorged mosquitoes was identified. The distance from the enclosure of the animal serving as a ... More
Deep ion sequencing of amplicon adapter ligated libraries: a novel tool in molecular diagnostics of formalin fixed and paraffin embedded tissues.
AuthorsBecker K, Vollbrecht C, Koitzsch U, Koenig K, Fassunke J, Huss S, Nuernberg P, Heukamp LC, Buettner R, Odenthal M, Altmueller J, Merkelbach-Bruse S
JournalJ Clin Pathol
PubMed ID23618693
Due to the advanced progress in personalised therapy concepts for non-small cell lung cancer (NSCLC), we applied the ion semiconductor sequencing (ISS) approach to molecular diagnosis of NSCLC, analysing a set of therapy relevant gene loci. DNA from macrodissected tumour samples of formalin fixed biopsies was used for PCR amplification ... More
Genome-wide analysis of EGR2/SOX10 binding in myelinating peripheral nerve.
AuthorsSrinivasan R, Sun G, Keles S, Jones EA, Jang SW, Krueger C, Moran JJ, Svaren J
JournalNucleic Acids Res
PubMed ID22492709
Myelin is essential for the rapidity of saltatory nerve conduction, and also provides trophic support for axons to prevent axonal degeneration. Two critical determinants of myelination are SOX10 and EGR2/KROX20. SOX10 is required for specification of Schwann cells from neural crest, and is required at every stage of Schwann cell ... More
Endogenous retroviruses function as species-specific enhancer elements in the placenta.
AuthorsChuong EB, Rumi MA, Soares MJ, Baker JC
JournalNat Genet
PubMed ID23396136
The mammalian placenta is remarkably distinct between species, suggesting a history of rapid evolutionary diversification. To gain insight into the molecular drivers of placental evolution, we compared biochemically predicted enhancers in mouse and rat trophoblast stem cells (TSCs) and found that species-specific enhancers are highly enriched for endogenous retroviruses (ERVs) ... More
Molecular and genetic characteristics of small ruminant lentiviruses in Slovenia.
AuthorsKuhar U, Barlic-Maganja D, Zadnik T, Grom J
JournalActa Vet Hung
PubMed ID23439298
Small ruminant lentiviruses (SRLV) are spread throughout the world, including Slovenia, where the first evidence of caprine arthritis encephalitis virus (CAEV) infection was found in 1996. This study was conducted to investigate the molecular and genetic characteristics of SRLV infection in Slovenia in order to classify our strains in relation ... More
Structural bias in T4 RNA ligase-mediated 3'-adapter ligation.
AuthorsZhuang F, Fuchs RT, Sun Z, Zheng Y, Robb GB
JournalNucleic Acids Res
PubMed ID22241775
T4 RNA ligases are commonly used to attach adapters to RNAs, but large differences in ligation efficiency make detection and quantitation problematic. We developed a ligation selection strategy using random RNAs in combination with high-throughput sequencing to gain insight into the differences in efficiency of ligating pre-adenylated DNA adapters to ... More
A novel dnaJ family gene, sflA, encodes an inhibitor of flagellation in marine Vibrio species.
AuthorsKitaoka M, Nishigaki T, Ihara K, Nishioka N, Kojima S, Homma M
JournalJ Bacteriol
PubMed ID23222726
The marine bacterium Vibrio alginolyticus has a single polar flagellum. Formation of that flagellum is regulated positively and negatively by FlhF and by FlhG, respectively. The ?flhF mutant makes no flagellum, whereas the ?flhFG double-deletion mutant usually lacks a flagellum. However, the ?flhFG mutant occasionally reverts to become motile by ... More
Two-step cross-linking for analysis of protein-chromatin interactions.
AuthorsTian B, Yang J, Brasier AR
JournalMethods Mol Biol
PubMed ID22113271
Eukaryotic gene regulation is controlled, in part, by inducible transcription factor-binding regulatory sequences in a tissue-specific and hormone-responsive manner. The development of methods for the analysis of transcription factor interaction within native chromatin has been a significant advance for the systematic analyses of the timing of gene regulation and studies ... More
Nrf2-MafG heterodimers contribute globally to antioxidant and metabolic networks.
AuthorsHirotsu Y, Katsuoka F, Funayama R, Nagashima T, Nishida Y, Nakayama K, Engel JD, Yamamoto M
JournalNucleic Acids Res
PubMed ID22965115
NF-E2-related factor 2 (Nrf2) is a key transcription factor that is critical for cellular defense against oxidative and xenobiotic insults. Nrf2 heterodimerizes with small Maf (sMaf) proteins and binds to antioxidant response elements (AREs) to activate a battery of cytoprotective genes. However, it remains unclear to what extent the Nrf2-sMaf ... More
Sequencing and annotated analysis of an Estonian human genome.
AuthorsLilleoja R, Sarapik A, Reimann E, Reemann P, Jaakma Ü, Vasar E, Kõks S
JournalGene
PubMed ID22138481
In present study we describe the sequencing and annotated analysis of the individual genome of Estonian. Using SOLID technology we generated 2,449,441,916 of 50-bp reads. The Bioscope version 1.3 was used for mapping and pairing of reads to the NCBI human genome reference (build 36, hg18). Bioscope enables also the ... More
Specific detection of naturally occurring hepatitis C virus mutants with resistance to telaprevir and boceprevir (protease inhibitors) among treatment-naïve infected individuals.
AuthorsFonseca-Coronado S, Escobar-Gutiérrez A, Ruiz-Tovar K, Cruz-Rivera MY, Rivera-Osorio P, Vazquez-Pichardo M, Carpio-Pedroza JC, Ruíz-Pacheco JA, Cazares F, Vaughan G
JournalJ Clin Microbiol
PubMed ID22116161
The use of telaprevir and boceprevir, both protease inhibitors (PI), as part of the specifically targeted antiviral therapy for hepatitis C (STAT-C) has significantly improved sustained virologic response (SVR) rates. However, different clinical studies have also identified several mutations associated with viral resistance to both PIs. In the absence of ... More
A probabilistic approach to learn chromatin architecture and accurate inference of the NF-?B/RelA regulatory network using ChIP-Seq.
AuthorsYang J, Mitra A, Dojer N, Fu S, Rowicka M, Brasier AR
Journal
PubMed ID23771139
Using nuclear factor-?B (NF-?B) ChIP-Seq data, we present a framework for iterative learning of regulatory networks. For every possible transcription factor-binding site (TFBS)-putatively regulated gene pair, the relative distance and orientation are calculated to learn which TFBSs are most likely to regulate a given gene. Weighted TFBS contributions to putative ... More
Electrofusion of mesenchymal stem cells and islet cells for diabetes therapy: a rat model.
AuthorsYanai G, Hayashi T, Zhi Q, Yang KC, Shirouzu Y, Shimabukuro T, Hiura A, Inoue K, Sumi S
Journal
PubMed ID23724055
Islet transplantation is a minimally invasive treatment for severe diabetes. However, it often requires multiple donors to accomplish insulin-independence and the long-term results are not yet satisfying. Therefore, novel ways to overcome these problems have been explored. Isolated islets are fragile and susceptible to pro-apoptotic factors and poorly proliferative. In ... More