Assessing the quality of the DNA sequence from the Human Genome Project.
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Base Sequence↗
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Large-scale comparison of protein sequence alignment algorithms with structure alignments.
Sequence alignment programs such as BLAST and PSI-BLAST are used routinely in pairwise, profile-based, or intermediate-sequence-search (ISS) methods to detect remote homologies for the purposes of fold assignment and comparative modeling. Yet, the sequence alignment quality of these methods at low sequence identity is not known. We have used the CE structure alignment program (Shindyalov and Bourne, Prot Eng 1998;11:739) to derive sequence alignments for all superfamily and family-level related proteins in the SCOP domain database. CE aligns structures and their sequences based on distances within each protein, rather than on interprotein distances. We compared BLAST, PSI-BLAST, CLUSTALW, and ISS alignments with the CE structural alignments. We found that global alignments with CLUSTALW were very poor at low sequence identity (<25%), as judged by the CE alignments. We used PSI-BLAST to search the nonredundant sequence database (nr) with every sequence in SCOP using up to four iterations. The resulting matrix was used to search a database of SCOP sequences. PSI-BLAST is only slightly better than BLAST in alignment accuracy on a per-residue basis, but PSI-BLAST matrix alignments are much longer than BLAST's, and so align correctly a larger fraction of the total number of aligned residues in the structure alignments. Any two SCOP sequences in the same superfamily that shared a hit or hits in the nr PSI-BLAST searches were identified as linked by the shared intermediate sequence. We examined the quality of the longest SCOP-query/ SCOP-hit alignment via an intermediate sequence, and found that ISS produced longer alignments than PSI-BLAST searches alone, of nearly comparable per-residue quality. At 10-15% sequence identity, BLAST correctly aligns 28%, PSI-BLAST 40%, and ISS 46% of residues according to the structure alignments. We also compared CE structure alignments with FSSP structure alignments generated by the DALI program. In contrast to the sequence methods, CE and structure alignments from the FSSP database identically align 75% of residue pairs at the 10-15% level of sequence identity, indicating that there is substantial room for improvement in these sequence alignment methods. BLAST produced alignments for 8% of the 10,665 nonimmunoglobulin SCOP superfamily sequence pairs (nearly all <25% sequence identity), PSI-BLAST matched 17% and the double-PSI-BLAST ISS method aligned 38% with E-values <10.0. The results indicate that intermediate sequences may be useful not only in fold assignment but also in achieving more complete sequence alignments for comparative modeling.
Algorithms↗
MRI of bowel obstruction using the HASTE sequence.
High quality MR images of the gastrointestinal tract are now obtainable using a T2-weighted fast SE MR sequence with half-Fourier acquisition of data (the HASTE sequence). This fast sequence can acquire a set of images in a single breath hold and is insensitive to peristalsis-related artifact. It can be used with other imaging modalities in defining bowel obstruction and is accurate in determining the presence of bowel dilation and level of obstruction and in differentiating obstructing tumor from other causes of obstruction.
Humans↗
Motion related artifacts in spin-echo MR imaging: effects of sequence parameters on image quality at 1.5 Tesla.
PURPOSE: The purpose of this study was to evaluate the effects of selected parameters in a conventional spin-echo MRI sequence on image quality in a phantom undergoing periodic motion. METHODS: Images of a composite oil/copper sulfate phantom moving in a 1.5 Tesla magnet were acquired. The CuSO4 ghost signal-to-noise ratio (SNR) and distances between corresponding points on the phantom image and adjacent ghosts were evaluated. RESULTS: Increasing time to repeat (TR) and number of acquisitions (NAq) improved image quality. Ghost SNR was near unity at TR = 500 msec and at NAq around 4. Increasing TR and NAq resulted in increased distance between images. The effects of field of view (FOV) and matrix size on image quality were relatively less significant. CONCLUSION: Increasing NAq appeared to have a comparatively greater effect on ghost SNR and image quality under our experimental conditions. However, the effect of increasing NAq on image quality appeared to be definite since a saturation point was reached after a particular NAq. Increasing NAq above this point resulted in a relatively smaller improvement in image quality.
Artifacts↗
Automatic assessment of alignment quality.
Multiple sequence alignments play a central role in the annotation of novel genomes. Given the biological and computational complexity of this task, the automatic generation of high-quality alignments remains challenging. Since multiple alignments are usually employed at the very start of data analysis pipelines, it is crucial to ensure high alignment quality. We describe a simple, yet elegant, solution to assess the biological accuracy of alignments automatically. Our approach is based on the comparison of several alignments of the same sequences. We introduce two functions to compare alignments: the average overlap score and the multiple overlap score. The former identifies difficult alignment cases by expressing the similarity among several alignments, while the latter estimates the biological correctness of individual alignments. We implemented both functions in the MUMSA program and demonstrate the overall robustness and accuracy of both functions on three large benchmark sets.
Algorithms↗
Improving the quality of automatic DNA sequence assembly using fluorescent trace-data classifications.
Virtually all large-scale sequencing projects use automatic sequence-assembly programs to aid in the determination of DNA sequences. The computer-generated assemblies required substantial hand-editing to transform them into submissions for GenBank. As the size of sequencing projects increases, it becomes essential to improve the quality of the automated assemblies so that this time consuming hand-editing may be reduced. Current ABI sequencing technology uses base calls made from fluorescently-labeled DNA fragments run on gels. We present a new representation for the fluorescent trace data associated with individual base calls. This representation can be used before, during, and after fragment assembly to improve the quality of assemblies. We demonstrate one such use-end-trimming of sub-optimal data-that results in a significant improvement in the quality of subsequent assemblies.
Algorithms↗
Primer design for automated DNA sequencing in a core facility.
We assessed the quality of nine standard primers for automated fluorescent dye terminator DNA sequencing by whether their melting temperatures (Tms) were in the optimal range for DNA sequencing, and the degree to which their sequences matched the sequences of 36 common vectors. The M13F (-21/-20), M13F (-41/-40), M13R, and T7R primers showed optimal physicochemical characteristics and were not redesigned. The M13R (-41/-40), T3, and SP6 primers showed mismatches and/or Tm values outside of the optimal range and were redesigned by these two criteria. With few exceptions, the redesigned primers did not significantly improve DNA sequencing quality as assessed by Phred scores compared to the corresponding original primer. However, both redesigned T7 primers, which were also redesigned to function as pET primers, strikingly improved sequencing on pET vectors. The original and redesigned T7 primers also improved the sequence quality for the Bluescript family of vectors compared to the original and redesigned M13R (-41/-40) primers. The mismatch-redesigned T7, SP6, and M13R (-41/-40) primers marginally improved the sequence quality over the corresponding original primers for specific vector types. Within limits, decreasing the %GC and Tm below reported optimal levels did not affect the sequence quality. Commercially available standard vector primers were surveyed.
Base Sequence↗
Methodologic European external quality assurance for DNA sequencing: the EQUALseq program.
BACKGROUND: DNA sequencing is a key technique in molecular diagnostics, but to date no comprehensive methodologic external quality assessment (EQA) programs have been instituted. Between 2003 and 2005, the European Union funded, as specific support actions, the EQUAL initiative to develop methodologic EQA schemes for genotyping (EQUALqual), quantitative PCR (EQUALquant), and sequencing (EQUALseq). Here we report on the results of the EQUALseq program. METHODS: The participating laboratories received a 4-sample set comprising 2 DNA plasmids, a PCR product, and a finished sequencing reaction to be analyzed. Data and information from detailed questionnaires were uploaded online and evaluated by use of a scoring system for technical skills and proficiency of data interpretation. RESULTS: Sixty laboratories from 21 European countries registered, and 43 participants (72%) returned data and samples. Capillary electrophoresis was the predominant platform (n = 39; 91%). The median contiguous correct sequence stretch was 527 nucleotides with considerable variation in quality of both primary data and data evaluation. The association between laboratory performance and the number of sequencing assays/year was statistically significant (P <0.05). Interestingly, more than 30% of participants neither added comments to their data nor made efforts to identify the gene sequences or mutational positions. CONCLUSIONS: Considerable variations exist even in a highly standardized methodology such as DNA sequencing. Methodologic EQAs are appropriate tools to uncover strengths and weaknesses in both technique and proficiency, and our results emphasize the need for mandatory EQAs. The results of EQUALseq should help improve the overall quality of molecular genetics findings obtained by DNA sequencing.
European Union↗
Sources of systematic error in functional annotation of genomes: domain rearrangement, non-orthologous gene displacement and operon disruption.
Functional annotation of proteins encoded in newly sequenced genomes can be expected to meet two conflicting objectives: (i) provide as much information as possible, and (ii) avoid erroneous functional assignments and over-predictions. The continuing exponential growth of the number of sequenced genomes makes the quality of sequence annotation a critical factor in the efforts to utilize this new information. When dubious functional assignments are used as a basis for subsequent predictions, they tend to proliferate, leading to "database explosion". It is therefore important to identify the common factors that hamper functional annotation. As a first step towards that goal, we have compared the annotations of the Mycoplasma genitalium and Methanococcus jannaschii genomes produced in several independent studies. The most common causes of questionable predictions appear to be: i) non-critical use of annotations from existing database entries; ii) taking into account only the annotation of the best database hit; iii) insufficient masking of low complexity regions (e.g. non-globular domains) in protein sequences, resulting in spurious database hits obscuring relevant ones; iv) ignoring multi-domain organization of the query proteins and/or the database hits; v) non-critical functional inferences on the basis of the functions of neighboring genes in an operon; vi) non-orthologous gene displacement, i.e. involvement of structurally unrelated proteins in the same function. These observations suggest that case by case validation of functional annotation by expert biologists remains crucial for productive genome analysis.
Databases, Factual↗
OligoSeq: Rapid nanopore-sequencing of single-stranded oligonucleotides.
Nanopore-based DNA sequencing technology has achieved remarkable success in sequencing increasingly long DNA strands (e.g., over a million nucleotides long) for genomics research and biotechnology applications. However, the same level of progress has not been achieved for DNA oligonucleotides (usually ≤ 300 nucleotides long). Oligonucleotides play a crucial role in genome engineering efforts through oligo library generation and in DNA data storage, where they are used to encode computer information, such as binary (digital) data in DNA libraries. To enable these applications, accurate sequencing of oligonucleotides in a way that allows to assess for sequence variability, quality and length is essential. But sequencing solutions for oligonucleotides - particularly DNA primers for PCR, oligo DNA libraries used for mutagenesis or cDNA libraries used in gene expression analysis - remain inadequate. To address this gap, OligoSeq is presented as an innovative approach that integrates two complementary techniques: AmpliSeq (based on PCR) and RevSeq (based on reverse complementation with sequence-specific or random primers) to facilitate sequencing of single-stranded oligonucleotides using reference sequence anchor matches of more than ≥ 90% identity spanning from about 70% to 10% with AmpliSeq or RevSeq with random nonamers, respectively, and resolving the final reference sequence based on the most likely candidate from basecall frequencies, regardless of length and double-stranding method. OligoSeq can be integrated with nanopore sequencing technology pipelines and can be used as a reference for other sequencing platforms requiring double-stranded adapters, offering a practical and scalable alternative for standard quality control in single-stranded oligonucleotide synthesis. The use of nanopore technology, compatible with the double-stranding methods showcased, is shown to be the most cost-effective method for resolving original DNA sequences of different length and quality, and to assess its sequence variability, compared to other methods such as Illumina, PacBio or HPLC/MS.
Sequence Analysis, DNA↗
RANDNA: a random DNA sequence generator.
Monte Carlo simulations are useful to verify the significance of data. Genomic regularities, such as the nucleotide correlations or the not uniform distribution of the motifs throughout genomic or mature mRNA sequences, exist and their significance can be checked by means of the Monte Carlo test. The test needs good quality random sequences in order to work, moreover they should have the same nucleotide distribution as the sequences in which the regularities have been found. Random DNA sequences are also useful to estimate the background score of an alignment, that is a threshold below which the resulting score is merely due to chance. We have developed RANDNA, a free software which allows to produce random DNA or RNA sequences setting both their length and the percentage of nucleotide composition. Sequences having the same nucleotide distribution of exonic, intronic or intergenic sequences can be generated. Its graphic interface makes it possible to easily set the parameters that characterize the sequences being produced and saved in a text format file. The pseudo-random number generator function of Borland Delphi 6 is used, since it guarantees a good randomness, a long cycle length and a high speed. We have checked the quality of sequences generated by the software, by means of well-known tests, both by themselves and versus genuine random sequences. We show the good quality of the generated sequences. The software, complete with examples and documentation, is freely available to users from: http://www.introni.it/en/software.
Base Sequence↗
Fast T2-weighted MR imaging: impact of variation in pulse sequence parameters on image quality and artifacts.
The purpose of this study was to quantitatively evaluate in a phantom model the practical impact of alteration of key imaging parameters on image quality and artifacts for the most commonly used fast T(2)-weighted MR sequences. These include fast spin-echo (FSE), single shot fast spin-echo (SSFSE), and spin-echo echo-planar imaging (EPI) pulse sequences. We developed a composite phantom with different T1 and T2 values, which was evaluated while stationary as well as during periodic motion. Experiments involved controlled variations in key parameters including effective TE, TR, echo spacing (ESP), receive bandwidth (BW), echo train length (ETL), and shot number (SN). Quantitative analysis consisted of signal-to-noise ratio (SNR), image nonuniformity, full-width-at-half-maximum (i.e., blurring or geometric distortion) and ghosting ratio. Among the fast T(2)-weighted sequences, EPI was most sensitive to alterations in imaging parameters. Among imaging parameters that we tested, effective TE, ETL, and shot number most prominently affected image quality and artifacts. Short T(2) objects were more sensitive to alterations in imaging parameters in terms of image quality and artifacts. Optimal clinical application of these fast T(2)-weighted imaging pulse sequences requires careful attention to selection of imaging parameters.
Artifacts↗
Mapping multiple co-sequenced T-DNA integration sites within the Arabidopsis genome.
MOTIVATION: Insertion mutagenesis, using transgenes or endogenous transposons, is a popular method for generating null mutations (knockouts) in model organisms. Insertions are mapped to specific genes by amplifying (via TAIL-PCR) and sequencing genomic regions flanking the inserted DNA. The presence of multiple TAIL-PCR templates in one sequencing reaction results in chimeric sequence of intermittently low quality. Standard processing of this sequence by applying Phred quality requirements results in loss of informative sequence, whereas not trimming low-quality sequence causes inclusion of low-complexity homopolymers from the ends of sequence runs. Accurate mapping of the flanking sequences is complicated by the presence of gene families. RESULTS: Methods for extracting informative regions from sequence traces obtained by sequencing multiple TAIL-PCR fragments in a single reaction are described. The completely sequenced Arabidopsis genome was used to identify informative TAIL-PCR sequence regions. Methods were devised to define and select high quality matches and precisely map each insert to the correct genome location. These methods were used to analyze sequence of TAIL-PCR-amplified flanking regions of the inserts from individual plants in a T-DNA-mutagenized population of Arabidopsis thaliana, and are applicable to similar situations where a reference genome can be used to extract information from poor-quality sequence.
Arabidopsis↗
The CLUSTAL_X windows interface: flexible strategies for multiple sequence alignment aided by quality analysis tools.
CLUSTAL X is a new windows interface for the widely-used progressive multiple sequence alignment program CLUSTAL W. The new system is easy to use, providing an integrated system for performing multiple sequence and profile alignments and analysing the results. CLUSTAL X displays the sequence alignment in a window on the screen. A versatile sequence colouring scheme allows the user to highlight conserved features in the alignment. Pull-down menus provide all the options required for traditional multiple sequence and profile alignment. New features include: the ability to cut-and-paste sequences to change the order of the alignment, selection of a subset of the sequences to be realigned, and selection of a sub-range of the alignment to be realigned and inserted back into the original alignment. Alignment quality analysis can be performed and low-scoring segments or exceptional residues can be highlighted. Quality analysis and realignment of selected residue ranges provide the user with a powerful tool to improve and refine difficult alignments and to trap errors in input sequences. CLUSTAL X has been compiled on SUN Solaris, IRIX5.3 on Silicon Graphics, Digital UNIX on DECstations, Microsoft Windows (32 bit) for PCs, Linux ELF for x86 PCs, and Macintosh PowerMac.
Algorithms↗
Assessing the quality of finished genomic sequence.
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Genome, Human↗
[MR cholangiopancreatography with single-shot fast-spin-echo sequences].
Imaging quality of MR cholangiopancreatography (MRCP) has recently made a great advance, and MRCP plays an important part in the diagnosis of pancreticobiliary diseases. To obtaining excellent-quality MRCP images, three conditions are required: fluid-to-background contrast, high spatial resolution, and suppression of respiratory motion artifacts. Respiratory motion artifacts, whose suppression is especially important for MRCP, can be controlled by the use of breath-holding, signal averaging, or respiratory triggering. These images are implemented in either single-slice technique or multi-slice technique. We have performed MRCP prior to endoscopic retrograde cholangiopancreatography (ERCP) in more than 100 patients. MRCP images were obtained as maximum-intensity-projection (MIP) reconstruction images and thick-slice projection images by using single-shot fast-spin-echo sequence, and MIP reconstruction images by using respiratory-triggered fast-spin-echo sequence. We reviewed MRCP imaging with single-shot fast-spin-echo sequences.
Aged↗
Application of HPLC-ESI-ITMS in the quality control of carboxyterminal sequence confirmation for the recombinant DNA product Hirudin Variant 3.
In the present study, HPLC-ESI-IT (ion-trap) MS was used for carboxyterminal (C-terminal) amino acid sequence confirmation of intact recombinant Hirudin Variant 3 (HV3) and alkylated HV3. The C-terminal amino acid sequence of HV3 was determined by the use of carboxypeptidase P (CPP), and by the combined use of carboxypeptidase P and carboxypeptidase Y (CPY). The C-terminal amino acid sequence of alkylated HV3 with 1,4-dithiothreitol (DTT) reduction was also confirmed by the combined use of CPP and CPY (abbreviated to CPP-CPY). Up to 19 amino acid residues were confirmed in the nanomolar concentration range by analyzing the molecular weights of the truncated peptides of HV3. Another five amino acids were confirmed in the nanomolar concentration range of alkylated HV3 with DTT reduction. For sequencing alkylated HV3 with DTT reduction, HV3 reduced with DTT followed by alkylation with iodoacetamide. The reaction mixture, which included alkylated HV3, DTT, and iodoacetamide, was then directly sequenced without any further pre-treatment. The reaction was designed in a time-, and concentration-dependent manner to obtain the maximum sequence information. The results showed that HPLC-ESI-ITMS cannot only determine the C-terminal amino acid sequence of HV3, but also gives important information about the enzymatic degradation and subsequent release of the C-terminal amino acids of HV3.
Alkylating Agents↗
Quality management of sequence-based methods for measuring HIV-1 drug resistance.
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Anti-HIV Agents↗