A rapid and reliable method for the purification of high-quality plasmid DNA for double-stranded sequencing.
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Base Sequence↗
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Worldwide evaluation of DNA sequencing approaches for identification of drug resistance mutations in the human immunodeficiency virus type 1 reverse transcriptase.
A panel (ENVA-1) of well-defined blinded samples containing wild-type and mutant human immunodeficiency virus type 1 (HIV-1) reverse transcriptase was analyzed by automated DNA sequencing in 23 laboratories worldwide. Drug resistance mutations at codons 41, 215, and 184 were present in the panel samples at different ratios to the wild type. The presence of mutant genotypes was determined qualitatively and quantitatively. All laboratories reported the presence of sequence heterogeneities at codons 41, 215, and 184 in one or more of the panel samples, though not all reported the correct codon genotypes. Two laboratories reported a mutant genotype in samples containing only the wild type, whereas two and three laboratories failed to detect the mutant genotypes at codons 41 and 215, respectively, in a completely mutant DNA population. Mutations present at relative concentrations of 25% of the total DNA population were successfully identified by 13 of 23, 10 of 23, and 16 of 23 labs for codons 41, 215, and 184Val, respectively. For more than 80% of those laboratories that qualitatively detected the presence of a mutation correctly, the estimated wild type/mutant ratio was less than 25% different from the input ratio in those samples containing 25 to 50% or 75% mutant input. This first multicenter study on the quality of DNA sequencing approaches for identifying HIV-1 drug resistance mutations revealed large interlaboratory differences in the quality of the results. The application of these procedures in their current state would in several cases lead to inaccurate or even incorrect diagnostic results. Therefore, proper quality control and standardization are urgently needed.
Codon↗
Diffusion-weighted imaging of the brain: comparison of stimulated- and spin-echo echo-planar sequences.
We used a rat model of focal cerebral ischaemia to compare stimulated-echo (STE) and spin-echo (SE) echo planar (EPI) diffusion-weighted sequences as regards image quality and accuracy of calculation of apparent diffusion coefficients (ADC). Focal cerebral ischaemia was induced by endovascular occlusion of the middle cerebral artery in five rats. MRI was performed on a 2.35 tesla imager. For diffusion-weighted imaging (DWI) we used STE-EPI and SE-EPI with different diffusion times (delta) of 15, 30, 45, 60, 75 and 90 ms using values of b of 200, 300, 400, 500, 600 and 700 s/mm2. We assessed image quality, the signal-to-noise-ratio (SNR) and the accuracy of the ADC calculated from both sequences. Infarcts were delineated in all cases, independent of sequence type and delta. The image quality and SNR of the SE-EPI images were significantly better, with a higher SNR than STE-EPI images for short and intermediate values of delta. However, when delta reached 75 ms STE-EPI became superior to SE-EPI. ADC calculated from STE-EPI images were smaller than those from SE-EPI images for short and intermediate diffusion times, possibly because of the lower SNR of the former. We suggest that SE-EPI sequences be used for DWI of the brain, particularly on experimental systems and whole-body imagers with enhanced gradient hardware, where it is possible to run highly diffusion-weighted sequences (b > 500 s/mm2) with delta less than 50 ms. However, when using very long values of delta because of hardware restrictions or for measurement of restricted diffusion, STE sequences give better results.
Animals↗
[Capillary array electrophoresis and high-throughput DNA sequencing].
Depending on the outputs of 10,080 DNA sequencing samples from the human genome project, technical features of capillary array electrophoresis are discussed. The results show that 88% of the total readlength were higher than 50 kbp/run. It means that most of the readlength could reach 500 bp or more with reasonable quality. The results were much better than expected when the technology of capillary array appeared about one year ago. A key parameter affecting the quality of DNA sequencing is the purity of sequencing template. Therefore, a batch agarose slab electrophoresis was applied to check the purity before loading. Other conditions, such as concentration of template and loading amount, are discussed as well. Finally, comparisons based on the electrophoresis timing, the pre-treatment of samples, the loading procedures and the results analysis between capillary array electrophoresis and slab gel electrophoresis have been made. From the point of view of high-throughput DNA sequencing, especially in the field of genome research, the capillary array electrophoresis should be the better choice as a more efficient technical platform.
Base Sequence↗
A custom library construction method for super-resolution ribosome profiling in Arabidopsis.
BACKGROUND: Ribosome profiling, also known as Ribo-seq, is a powerful technique to study genome-wide mRNA translation. It reveals the precise positions and quantification of ribosomes on mRNAs through deep sequencing of ribosome footprints. We previously optimized the resolution of this technique in plants. However, several key reagents in our original method have been discontinued, and thus, there is an urgent need to establish an alternative protocol. RESULTS: Here we describe a step-by-step protocol that combines our optimized ribosome footprinting in plants with available custom library construction methods established in yeast and bacteria. We tested this protocol in 7-day-old Arabidopsis seedlings and evaluated the quality of the sequencing data regarding ribosome footprint length, mapped genomic features, and the periodic properties corresponding to actively translating ribosomes through open resource bioinformatic tools. We successfully generated high-quality Ribo-seq data comparable with our original method. CONCLUSIONS: We established a custom library construction method for super-resolution Ribo-seq in Arabidopsis. The experimental protocol and bioinformatic pipeline should be readily applicable to other plant tissues and species.
3-nt periodicity↗
[Magnetic resonance tomography in the diagnosis of mandibular osteomyelitis].
PURPOSE: A prospective study of the value of MRI in the diagnosis of osteomyelitis of the mandible with special reference to the suitability of different MR sequences. MATERIAL AND METHODS: In 13 patients, average age 55 years (12-82), with clinical suspicion of osteomyelitis of the mandible, 18 MRI examinations were carried out (STIR, TSE T2, proton and SE T1 weighted scans with and without contrast, slice thickness 3.5 to 6 mm). Image quality of the sequences was evaluated as well as the suitability of the various sequences for showing the lesion, its location and extent. Activity of the osteomyelitis was judged by the degree of contrast uptake and was correlated with 3-phase bone scintigraphy and with histological findings. RESULTS: In 9 of the 14 cases the findings on MRI and of the scintigraphy agreed with the histology. In two patients the activity of the inflammatory process was exaggerated by the MRI. In another follow-up examination it was slightly underestimated. All lesions were shown to be highly active by the histology were recognized as such by MRI. For the localisation and recognition of the extent of the inflammatory processes STIR sequences and T1 weighted non-enhanced SE sequences proved the most suitable. Contrast medium is essential to evaluate the inflammatory activity. CONCLUSION: MRI is a sensitive diagnostic method; it is as good as 3-phase bone scintigraphy in demonstrating osteomyelitis of the mandible and of its activity but is superior for showing the pathological anatomy.
Adolescent↗
Standardized volume-rendering of contrast-enhanced renal magnetic resonance angiography.
PURPOSE: To propose a technique for standardizing volume-rendering technique (VRT) protocols and to compare this with maximum intensity projection (MIP) in regard to image quality and diagnostic confidence in stenosis diagnosis with magnetic resonance angiography (MRA). MATERIAL AND METHODS: Twenty patients were examined with MRA under suspicion of renal artery stenosis. Using the histogram function in the volume-rendering software, the 95th and 99th percentiles of the 3D data set were identified and used to define the VRT transfer function. Two radiologists assessed the stenosis pathology and image quality from rotational sequences of MIP and VRT images. RESULTS: Good overall agreement (mean kappa=0.72) was found between MIP and VRT diagnoses. The agreement between MIP and VRT was considerably better than that between observers (mean kappa=0.43). One of the observers judged VRT images as having higher image quality than MIP images. CONCLUSION: Presenting renal MRA images with VRT gave results in good agreement with MIP. With VRT protocols defined from the histogram of the image, the lack of an absolute gray scale in MRI need not be a major problem.
Adult↗
Mitochondrial genes and mammalian phylogenies: increasing the reliability of branch length estimation.
Since branch lengths provide important information about the timing and the extent of evolutionary divergence among taxa, accurate resolution of evolutionary history depends as much on branch length estimates as on recovery of the correct topology. However, the empirical relationship between the choice of genes to sequence and the quality of branch length estimation remains ill defined. To address this issue, we evaluated the accuracy of branch lengths estimated from subsets of the mitochondrial genome for a mammalian phylogeny with known subordinal relationships. Using maximum-likelihood methods, we estimated branch lengths from an 11-kb sequence of all 13 protein-coding genes and compared them with estimates from single genes (0.2-1.8 kb) and from 7 different combinations of genes (2-3.5 kb). For each sequence, we separated the component of the log-likelihood deviation due to branch length differences associated with alternative topologies from that due to those that are independent of the topology. Even among the sequences that recovered the same tree topology, some produced significantly better branch length estimates than others did. The combination of correct topology and significantly better branch length estimation suggests that these gene combinations may prove useful in estimating phylogenetic relationships for mammalian divergences below the ordinal level. Thus, the proper choice of genes to sequence is a critical factor for reliable estimation of evolutionary history from molecular data.
Animals↗
MR rectography evaluation of rectoceles with oral gadopentetate dimeglumine and polyethylene glycol solution.
BACKGROUND: We assessed the value of magnetic resonance (MR) rectography in diagnosing rectocele by using a combination of oral gadopentetate dimeglumine and polyethylene glycol solutions. METHODS: T1-weighted, breath-hold, fast low-angle shot sequences were made in 22 patients in resting and straining states in the supine position before and after administration of oral paramagnetic contrast. Twelve patients received polyethylene glycol solution in addition to contrast, and 10 received contrast only. The pubococcygeal line was the reference used for diagnostic measurements. The quality of each sequence was graded. Paired t test, chi-square test, and the Kolmogorov-Smirnov test were applied for statistical analysis. RESULTS: The results of noncontrast images were inconclusive. There was a statistically significant difference between MR imaging diagnosis of rectocele in patients in the straining position without and with contrast ( p < 0.05), and the difference was more prominent in patients using polyethylene glycol solution ( p < 0.001). Using oral contrast plus polyethylene glycol produced significantly better MR examinations in the resting and straining positions ( p < 0.05). CONCLUSION: The combination of oral contrast and polyethylene glycol solution improves the diagnostic value of MR rectography and is particularly useful when noncontrast examinations are inconclusive.
Administration, Oral↗
Transcriptome analysis of channel catfish (Ictalurus punctatus): initial analysis of gene expression and microsatellite-containing cDNAs in the skin.
Previous molecular genetic studies on channel catfish (Ictalurus punctatus) have focused on limited number of genes and gene products. Recent advancement of molecular techniques made high throughput analysis of transcriptomes possible. As part of our transcriptome analysis of channel catfish, we have analyzed 1909 expressed sequence tags (ESTs) derived from a skin library. Of the 1909 ESTs analyzed, 1376 (72.1%) ESTs representing 496 unique genes had homologies with other organisms while 478 (25.0%) ESTs had no significant homologies and were designated as unknown. The remaining 55 (2.9%) EST clones were eliminated because of their low quality or short sequences. Of the 496 unique genes, 327 (65.9%) genes were singletons while 169 (34.1%) genes represented by two or more ESTs. A total of 1007 (52.8%) ESTs representing 235 unique genes matched previously reported channel catfish ESTs while 847 (44.4%) ESTs representing 261 unique genes were newly identified from this research. Functional categorization of the channel catfish genes indicated that the largest group was ribosomal proteins with 65 unique genes represented by 500 clones. The most abundantly expressed gene, the calcium binding protein ictacalcin, accounted for almost 5% of overall expression, indicating its important function in the skin. Sequence analysis of ESTs revealed the presence of 89 microsatellite-containing genes that may be valuable for future mapping studies.
Animals↗
Model building by comparison: a combination of expert knowledge and computer automation.
The CASP blinds trials (Critical Assessment of techniques for protein Structure Prediction) assess the accuracy of protein prediction that includes evaluation of comparative model building of protein structures. Comparative models of four proteins (T0001, T0003, T0017, and T0028) for CASP2 (held during 1996) were constructed using computer algorithms combined with visual inspection. Essentially the main-chain modelling involves construction of the target structure from rigid-body segments of homologues and loop fragments extracted from homologous and nonredundant databases. Side-chains were initially constructed by inheritance from the parent or from a rotamer library. Side-chain conformations were then refined using a novel mean field approach that includes solvation. Comparison of the models with the subsequently released X-ray structures identified the successes and limitations of our approach. The most problematic area is the quality of the sequence alignments between parent(s) and target. In this respect the overinterpretation of the conserved features within homologous families can be misleading. Several features of our approach have a positive effect on the accuracy of the models. For T0003, inspection correctly identified that a lower sequence identity parent provides the best framework for this model. Loop selection worked well where a homologous protein fragment was used, but that the use of nonredundant fragment library remains problematic for hinge movements and displacements in secondary structure elements relative to the parent. Side-chain refinement improved residue conformations relative to the initial model. Use of limited energy minimization improved the stereochemical quality of the model without increasing the RMS deviation. This study has identified methods that are effective and areas requiring further attention to improve model building by comparison.
Amino Acid Sequence↗
MR CAT scan: a modular approach for hybrid imaging.
In this study, a modular concept for NMR hybrid imaging is presented. This concept essentially integrates different imaging modules in a sequential fashion and is therefore called CAT (combined acquisition technique). CAT is not a single specific measurement sequence, but rather a sequence design concept whereby distinct acquisition techniques with varying imaging parameters are employed in rapid succession in order to cover k-space. The power of the CAT approach is that it provides a high flexibility toward the acquisition optimization with respect to the available imaging time and the desired image quality. Important CAT sequence optimization steps include the appropriate choice of the k-space coverage ratio and the application of mixed bandwidth technology. Details of both the CAT methodology and possible CAT acquisition strategies, such as FLASH/EPI-, RARE/EPI- and FLASH/BURST-CAT are provided. Examples from imaging experiments in phantoms and healthy volunteers including mixed bandwidth acquisitions are provided to demonstrate the feasibility of the proposed CAT concept.
Abdomen↗
MIPS bacterial genomes functional annotation benchmark dataset.
MOTIVATION: Any development of new methods for automatic functional annotation of proteins according to their sequences requires high-quality data (as benchmark) as well as tedious preparatory work to generate sequence parameters required as input data for the machine learning methods. Different program settings and incompatible protocols make a comparison of the analyzed methods difficult. RESULTS: The MIPS Bacterial Functional Annotation Benchmark dataset (MIPS-BFAB) is a new, high-quality resource comprising four bacterial genomes manually annotated according to the MIPS functional catalogue (FunCat). These resources include precalculated sequence parameters, such as sequence similarity scores, InterPro domain composition and other parameters that could be used to develop and benchmark methods for functional annotation of bacterial protein sequences. These data are provided in XML format and can be used by scientists who are not necessarily experts in genome annotation. AVAILABILITY: BFAB is available at http://mips.gsf.de/proj/bfab
Bacterial Proteins↗
Cancer and genomics.
Identification of the genes that cause oncogenesis is a central aim of cancer research. We searched the proteins predicted from the draft human genome sequence for paralogues of known tumour suppressor genes, but no novel genes were identified. We then assessed whether it was possible to search directly for oncogenic sequence changes in cancer cells by comparing cancer genome sequences against the draft genome. Apparently chimaeric transcripts (from oncogenic fusion genes generated by chromosomal translocations, the ends of which mapped to different genomic locations) were detected to the same degree in both normal and neoplastic tissues, indicating a significant level of false positives. Our experiment underscores the limited amount and variable quality of DNA sequence from cancer cells that is currently available.
Base Sequence↗
All known in vivo functions of the Oct-2 transcription factor require the C-terminal protein domain.
Oct-2, a transcription factor expressed in the B lymphocyte lineage and in the developing CNS, functions through of a number of discrete protein domains. These include a DNA-binding POU homeodomain flanked by two transcriptional activation domains. In vitro studies have shown that the C-terminal activation domain, a serine-, threonine- and proline-rich sequence, possesses unique qualities, including the ability to activate transcription from a distance in a B cell-specific manner. In this study, we describe mice in which the endogenous oct-2 gene has been modified through gene targeting to create a mutated allele, oct-2DeltaC, which encodes Oct-2 protein isoforms that lack all sequence C-terminal to the DNA-binding domain. Surprisingly, despite the retention of the DNA-binding domain and the glutamine-rich N-terminal activation domain, the truncated protein(s) encoded by the oct-2DeltaC allele are unable to rescue any of the previously described defects exhibited by oct-2 null mice. Homozygous oct-2DeltaC/DeltaC mice die shortly after birth, and B cell maturation, B-1 cell self renewal, serum Ig levels, and B lymphocyte responses to in vitro stimulation are all reduced or absent, to a degree equivalent to that seen in oct-2 null mice. We conclude that the C-terminal activation domain of Oct-2 is required to mediate the unique and indispensable functions of the Oct-2 transcription factor in vivo.
Amino Acid Sequence↗
Gene discovery using computational and microarray analysis of transcription in the Drosophila melanogaster testis.
Identification and annotation of all the genes in the sequenced Drosophila genome is a work in progress. Wild-type testis function requires many genes and is thus of potentially high value for the identification of transcription units. We therefore undertook a survey of the repertoire of genes expressed in the Drosophila testis by computational and microarray analysis. We generated 3141 high-quality testis expressed sequence tags (ESTs). Testis ESTs computationally collapsed into 1560 cDNA set used for further analysis. Of those, 11% correspond to named genes, and 33% provide biological evidence for a predicted gene. A surprising 47% fail to align with existing ESTs and 16% with predicted genes in the current genome release. EST frequency and microarray expression profiles indicate that the testis mRNA population is highly complex and shows an extended range of transcript abundance. Furthermore, >80% of the genes expressed in the testis showed onefold overexpression relative to ovaries, or gonadectomized flies. Additionally, >3% showed more than threefold overexpression at p <0.05. Surprisingly, 22% of the genes most highly overexpressed in testis match Drosophila genomic sequence, but not predicted genes. These data strongly support the idea that sequencing additional cDNA libraries from defined tissues, such as testis, will be important tools for refined annotation of the Drosophila genome. Additionally, these data suggest that the number of genes in Drosophila will significantly exceed the conservative estimate of 13,601.
Animals↗
Identification of target genes regulated by FOXC1 using nickel agarose-based chromatin enrichment.
PURPOSE: To overcome the problem of antibody availability, often encountered during chromatin immunoprecipitation (ChIP) assays, nickel agarose-based chromatin enrichment (NACE) was developed. Based on the affinity of (His)-6-tagged proteins for the nickel ion, this modified form of ChIP allows the isolation of chromatin in the absence of specific antibodies. METHODS: Nonpigmented ciliary epithelium cells were transfected with (His)-6-tagged FOXC1. FOXC1-enriched chromatin complexes were isolated by using the tight electrostatic interaction between histidine residues of the recombinant FOXC1 protein and nickel. One hundred fifty NACE-enriched clones were sequenced and subjected to in silico and biochemical analyses. RESULTS: Twenty-six clones were detected near known genes: Eight were near predicted but uncharacterized genes, eight were within areas where neither known nor predicted genes have yet been mapped, four were chimeric, and the rest were either repetitive (n=81) or poor-quality (n=23) sequences. Twenty of the 26 known genes were expressed in the eye. Five of the NACE-enriched clones (BMP2K, DACH, FVT-1, SIX-1, and PGE-2 receptor), as well as nine clones selected from the literature, were validated by PCR amplification in two independent lots of NACE-enriched chromatin. All five NACE-selected genes were detected in two independent assays, as well as four (BMP7, SMAD2, TGF-B1, and WNT6) of the nine genes selected from the literature, consistent with these genes' being regulated by FOXC1. CONCLUSIONS: NACE is a useful technique allowing specific chromatin enrichment in cases where antibodies are unavailable. Specific recovery of PTGER, DACH1, WNT6, and FVT-1 implicates FOXC1 in a variety of cellular events including modulation of intraocular pressure, cell cycle, ocular development, and oncogenesis.
Chromatin↗
Theoretical studies of the selective binding to DNA of two non-intercalating ligands: netropsin and SN 18071.
A theoretical study of the binding to DNA of netropsin and a bisquaternary ammonium heterocycle, SN 18071, is undertaken with an energy minimizing program based on empirical potential functions. The positioning of the ligand is achieved by force and torque calculations and its internal flexibility is taken into account. The binding preference of both drugs studied for the AT minor groove of B-DNA is shown to depend on both the electrostatic potential generated by the base sequence and the quality of the steric fit of the ligand in the groove. Ligand-DNA hydrogen bonds are shown to aid binding, but not to be essential in establishing binding preferences.
Amino Acid Sequence↗