E. coli host strains significantly affect the quality of small scale plasmid DNA preparations used for sequencing.
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Bacterial Proteins↗
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Results for “Sequencing quality”
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Keeping genome databases clean and up to date.
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Base Sequence↗
Pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of Plasmodium vivax in human patients.
BACKGROUND: Plasmodium vivax is the most widely distributed human malaria, responsible for 70-80 million clinical cases each year and large socio-economical burdens for countries such as Brazil where it is the most prevalent species. Unfortunately, due to the impossibility of growing this parasite in continuous in vitro culture, research on P. vivax remains largely neglected. METHODS: A pilot survey of expressed sequence tags (ESTs) from the asexual blood stages of P. vivax was performed. To do so, 1,184 clones from a cDNA library constructed with parasites obtained from 10 different human patients in the Brazilian Amazon were sequenced. Sequences were automatedly processed to remove contaminants and low quality reads. A total of 806 sequences with an average length of 586 bp met such criteria and their clustering revealed 666 distinct events. The consensus sequence of each cluster and the unique sequences of the singlets were used in similarity searches against different databases that included P. vivax, Plasmodium falciparum, Plasmodium yoelii, Plasmodium knowlesi, Apicomplexa and the GenBank non-redundant database. An E-value of <10(-30) was used to define a significant database match. ESTs were manually assigned a gene ontology (GO) terminology RESULTS: A total of 769 ESTs could be assigned a putative identity based upon sequence similarity to known proteins in GenBank. Moreover, 292 ESTs were annotated and a GO terminology was assigned to 164 of them. CONCLUSION: These are the first ESTs reported for P. vivax and, as such, they represent a valuable resource to assist in the annotation of the P. vivax genome currently being sequenced. Moreover, since the GC-content of the P. vivax genome is strikingly different from that of P. falciparum, these ESTs will help in the validation of gene predictions for P. vivax and to create a gene index of this malaria parasite.
AT Rich Sequence↗
Documenting ancient DNA quality via alpha satellite amplification and assessment of clone sequence diversity.
C/G-->T/A nucleotide alterations have been shown to hamper the straightforward interpretation of mitochondrial DNA sequence data derived from ancient tissues. Attempting to characterise this finding with respect to nuclear DNA, we contrasted two established protocols: (i) an enzymatic repair of damaged DNA, thereby translating and closing nicks in the DNA, and (ii) the application of N-phenacylthiazolium bromide, which cleaves glucose-derived protein crosslinks, presumably derived from Maillard reactions. We used medieval human bones that were refractory to standard PCR procedures. Due to negligible presence of short tandem repeat loci and also mitochondrial sequences, the extracted ancient DNA needed a higher copy PCR system to yield amplification products. The chosen PCR target was specific alphoid repetitive DNA with an experimentally determined minimum of 1000 copies per haploid genome. Alphoid repeat segments were generated from both contemporary DNA and DNA extracts of two human skeletons dating from 450-600 AD (omitting uracil N-glycosylase pre-treatment of the extracted samples), and were subsequently cloned and sequenced. The sequences were evaluated for the number and type of nucleotide alterations noted after the different pre-treatments, and were compared to our alphoid consensus sequence generated from modern DNA. Both methods failed to reflect the expected 32% variability among single alphoid repeats (accounting for locus-specific differences and polymerase errors) as well as to display the actual 2.88 ratio of transitions to transversions. Our data obtained from high-copy-number nuclear DNA mirror the phenomenon of sequence deviations observed in mitochondrial DNA extracted from old specimens.
Base Sequence↗
Construction of Human Immunoglobulin Combinatorial Library and Screening of Phage Antibodies to Hepatitis B Surface Antigen.
Human immunoglobulin combinatorial library was generated by using phage surface-display expression system, and phage antibodies (Fab fragments) to hepatitis B surface antigen (HbsAg) were screened from it. The products by half-nested PCR using signal peptide sequences as primers were superior in quality and quantity to those by PCR with conserved sequences in the 5'-end variable regions as primers. After three round of selections by biopanning, the ratio of positive clone was 69%. The inhibition assay showed the phage antibodies to be specifically anti-HbsAg. The V(H) genes were derived from V(H) I and V(H) III, while V(L)s belonged to V(lambda) II and V(lambda) I as shown by DNA sequencing.
Journal Article↗
[Well-balanced material sequences--principles and assumptions in the manufacture of high quality prostheses].
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Dental Materials↗
A genome sequence survey of the mollicute corn stunt spiroplasma Spiroplasma kunkelii.
The mollicute corn stunt spiroplasma (Spiroplasma kunkelii) is a leafhopper-transmitted pathogen of maize. Sequencing of the approximately 1.6-Mb genome of S. kunkelii was initiated to aid understanding the genetic basis of spiroplasma interactions with their plant and leafhopper hosts. In total, 144712 nucleotides of non-redundant, high-quality S. kunkelii genome sequence were obtained. Sequence tags were searched against the Mycoplasmataceae and Bacillus/Clostridium databases. Results showed that, in addition to spiroplasma phage SpV1 DNA insertions, spiroplasma genomes harbor more purine and amino acid biosynthesis, transcription regulation, cell envelope and DNA transport/binding genes than Mycoplasmataceae genomes. This investigation demonstrates that survey sequencing is an efficient procedure for gene discovery and genome characterization. The results of the S. kunkelii sequencing project are available at the Spiroplasma WebPage at http://www.oardc.ohio-state.edu/spiroplasma/genome.htm.
DNA Replication↗
Comparison of 3D segmented gradient-echo and steady-state free precession coronary MRI sequences in patients with coronary artery disease.
OBJECTIVE: Our objective was to compare two state-of-the-art coronary MRI (CMRI) sequences with regard to image quality and diagnostic accuracy for the detection of coronary artery disease (CAD). SUBJECTS AND METHODS: Twenty patients with known CAD were examined with a navigator-gated and corrected free-breathing 3D segmented gradient-echo (turbo field-echo) CMRI sequence and a steady-state free precession sequence (balanced turbo field-echo). CMRI was performed in a transverse plane for the left coronary artery and a double-oblique plane for the right coronary artery system. Subjective image quality (1- to 4-point scale, with 1 indicating excellent quality) and objective image quality parameters were independently determined for both sequences. Sensitivity, specificity, and accuracy for the detection of significant (> or = 50% diameter) coronary artery stenoses were determined as defined in invasive catheter X-ray coronary angiography. RESULTS: Subjective image quality was superior for the balanced turbo field-echo approach (1.8 +/- 0.9 vs 2.3 +/- 1.0 for turbo field-echo; p < 0.001). Vessel sharpness, signal-to-noise ratio, and contrast-to-noise ratio were all superior for the balanced turbo field-echo approach (p < 0.01 for signal-to-noise ratio and contrast-to-noise ratio). Of the 103 segments, 18% of turbo field-echo segments and 9% of balanced turbo field-echo segments had to be excluded from disease evaluation because of insufficient image quality. Sensitivity, specificity, and accuracy for the detection of significant coronary artery stenoses in the evaluated segments were 92%, 67%, 85%, respectively, for turbo field-echo and 82%, 82%, 81%, respectively, for balanced turbo field-echo. CONCLUSION: Balanced turbo field-echo offers improved image quality with significantly fewer nondiagnostic segments when compared with turbo field-echo. For the detection of CAD, both sequences showed comparable accuracy for the visualized segments.
Aged↗
Improving protein secondary structure prediction with aligned homologous sequences.
Most recent protein secondary structure prediction methods use sequence alignments to improve the prediction quality. We investigate the relationship between the location of secondary structural elements, gaps, and variable residue positions in multiple sequence alignments. We further investigate how these relationships compare with those found in structurally aligned protein families. We show how such associations may be used to improve the quality of prediction of the secondary structure elements, using the Quadratic-Logistic method with profiles. Furthermore, we analyze the extent to which the number of homologous sequences influences the quality of prediction. The analysis of variable residue positions shows that surprisingly, helical regions exhibit greater variability than do coil regions, which are generally thought to be the most common secondary structure elements in loops. However, the correlation between variability and the presence of helices does not significantly improve prediction quality. Gaps are a distinct signal for coil regions. Increasing the coil propensity for those residues occurring in gap regions enhances the overall prediction quality. Prediction accuracy increases initially with the number of homologues, but changes negligibly as the number of homologues exceeds about 14. The alignment quality affects the prediction more than other factors, hence a careful selection and alignment of even a small number of homologues can lead to significant improvements in prediction accuracy.
Amino Acid Sequence↗
Evaluation of the anatomical and functional properties of deglutition with various kinetic high-speed MRI sequences.
We evaluated various fast MR sequences for obtaining anatomical and dynamic functional information during deglutition. Seven healthy volunteers underwent MRI of the oropharynx during swallowing of an oral positive-contrast agent. Single-slice imaging was performed in the median sagittal plane while subjects were in a supine position. Twenty serial images were obtained using EPI, FLASH, and turbo-FLASH sequences. The dynamic (movement-related) information and the anatomical resolution of the soft tissues were evaluated during deglutition. The FLASH sequence provided high-quality images at rest. During swallowing, however, the images were significantly degraded by movement artifacts and had inferior temporal resolution. The EPI evidenced better temporal resolution, but was degraded by strong distortions and movement artifacts. The turbo-FLASH sequence provided the best temporal resolution and sufficient spatial resolution during motion. This sequence proved optimal for the investigation of swallowing function, and is expected to be of value for the documentation of functional disturbances in patients with oropharyngeal pathology.
Deglutition↗
A quantitative method for fast diffusion imaging using magnetization-prepared TurboFLASH.
For the in vivo measurement of the apparent diffusion coefficient (ADC), it is desirable for the total imaging time to be as short as possible. One technique is based on a TurboFLASH acquisition in which the diffusion gradients are inserted into a driven equilibrium Fourier transform (DEFT) combination of hard pulses. However, this sequence has the disadvantage that eddy current-induced inhomogeneities lead to incomplete refocusing of the magnetization during the diffusion preparation and to incorrect ADC values. A modification to the sequence is suggested that eliminates this error by phase-cycling the second 90 degrees pulse of the preparation. This study also investigates the effect of a reduced delay time between acquisitions on the accuracy of the measurement. The quality of the TurboFLASH sequence is demonstrated by experimental validation on an agar phantom and in vivo on the rat brain using a high-field (8.5 T) system. Reduction of the interexperiment delay time is shown to be achievable to a certain degree without compromising the measurement accuracy.
Animals↗
A comparison of MR sequences for lesions of the parotid gland.
PURPOSE: To compare six MR sequences (plain and gadolinium-enhanced fat suppressed T1-weighted spin echo, T2-weighted standard spin echo, fat-suppressed and non-fat-suppressed T2-weighted fast spin echo, and inversion-recovery T2-weighted fast spin echo) in their ability to detect, delineate, and characterize lesions of the parotid gland. METHODS: Fifty-eight parotid gland lesions imaged on 47 examinations were retrospectively evaluated by three blinded observers. Several outcome-related variables were compared by the above six sequences: imaging time, image quality, anatomic sharpness of parotid space, subjective lesion conspicuity, detected abnormality volume, number of individual lesions or discrete lobulations, conspicuity of invasion into adjacent boundaries and structures, and overall diagnostic value. RESULTS: Differences in the above outcome variables between sequences did not correlate with MR scanner software upgrade level, coil type, or lesion-dependent characteristics. Fat-suppressed fast spin-echo T2-weighted and inversion-recovery fast spin-echo T2-weighted sequences resulted in significantly higher scores for lesion conspicuity, detected abnormality volume, and overall diagnostic value. T1-weighted images resulted in the next highest scores, whereas gadolinium-enhanced T1-weighted and standard spin-echo T2-weighted sequences performed poorly for most parotid lesions. CONCLUSION: MR imaging of the parotid gland should include fat-suppressed, long-repetition-time, fast spin-echo T2-weighted, and T1-weighted sequences. Gadolinium-enhanced images need not be obtained routinely.
Adolescent↗
[MR cisternography using "long echo train length fast spin echo sequence" for demonstrating the inner ear].
Excellent quality of MR cisternography was acquired using "long echo train length fast spin echo sequence" (TR/TE = 2666/200, ETL = 24, 6 or 8 NEX, 3 mm thickness, 0 mm interslice gap, 19 cm FOV, 512 * 384, 2 DFT method). The inner ear anatomy such as canaliculus cochleae or lamina spiralis ossea were well visualized. The VII, VIII th nerve bundles within the internal auditory canal were detectable as 1 to 4 bundles. The vessels in the cerebellopontine angle or the internal auditory canal were also demarcated from the VII, VIII th nerve bundles because of their flow void. Signal to noise ratio seemed to be better than 3 DFT method, however limited spatial resolution in the cranio-caudal direction might require additional sagittal or coronal scan.
Adult↗
Escherichia coli spotted double-strand DNA microarrays: RNA extraction, labeling, hybridization, quality control, and data management.
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DNA, Complementary↗
A hot alkaline plasmid DNA miniprep method for automated DNA sequencing protocols.
A hot alkaline lysis procedure for plasmid DNA isolation is described for the preparation of high-quality substrates for automated DNA sequence analysis. This miniprep procedure employs routine laboratory chemicals while avoiding the use of enzymes and organic solvents to produce plasmid DNA free of RNA contamination. The method is inexpensive, relatively quick and can yield over 20 micrograms plasmid DNA from a 5-ml overnight culture. Automated sequencing has a success rate of > or = 96% with plasmid substrates prepared by this method. In addition, the plasmid DNAs can be used successfully in all types of molecular biology manipulations.
DNA, Bacterial↗
Should non-peer-reviewed raw DNA sequence data release be forced on the scientific community?
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Base Sequence↗
Prospective comparison of fast SE and GRASE sequences and echo planar imaging with conventional SE sequences in the detection of focal liver lesions at 1.0 T.
PURPOSE: Our goal was to investigate the value of T2-weighted fast SE (FSE) sequences, FSE sequences with shortened echo spacing (UFSE), combined gradient and spin echo sequences (GRASE), and segmented T1-weighted echo planar imaging (EPI) in comparison with conventional SE sequences in the detection of focal liver lesions. METHOD: Thirty-five patients with malignant focal liver lesions underwent MRI at 1.0 T. RESULTS: All fast T2-weighted imaging techniques (FSE, UFSE, GRASE) showed fewer artifacts and overall better image quality than the conventional SE sequence. Quantitative analysis demonstrated that UFSE imaging had the highest tumor/liver contrast/noise ratio (C/N) (13.9 +/- 5.5) followed by FSE (12.7 +/- 4.5), T2 SE (10.9 +/- 4.2), and GRASE (10.0 +/- 4.8) sequences. The differences in C/N between the UFSE and T2 SE sequences was statistically significant (p < 0.05). C/N was significantly higher (p < 0.01) for the T1 SE (-7.4 +/- 3.8) than for the EPI sequence (-0.5 +/- 5.6). CONCLUSION: FSE sequences with shortened echo spacing are valuable for liver imaging at 1.0 T. In comparison with the T2 SE sequence, they yielded better image quality and comparable tumor/liver C/N.
Adult↗
[Quality plan of a bone tissue bank].
In 1994, the French bioethics laws changed the regulations concerning donated human tissues with safety precautions and therapeutic use. The decree of 29(th) December 1998 describes the practical obligations required of bone tissue banks for approval by the French Health Ministry. Before the new regulations, a quality system was implemented at the Cochin hospital bone tissue bank. It integrates quality control for each step of the general process. We describe the quality plan of the Cochin Hospital bone tissue bank and show that the specific quality practices, resources and sequence of activities improve distribution of human bone allografts while maintaining their traceability.
Bioethics↗