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PubMed1996

A graph theoretic approach to the analysis of DNA sequencing data.

The analysis of data from automated DNA sequencing instruments has been a limiting factor in the development of new sequencing technology. A new base-calling algorithm that is intended to be independent of any particular sequencing technology has been developed and shown to be effective with data from the Applied Biosystems 373 sequencing system. This algorithm makes use of a nonlinear deconvolution filter to detect likely oligomer events and a graph theoretic editing strategy to find the subset of those events that is most likely to correspond to the correct sequence. Metrics evaluating the quality and accuracy of the resulting sequence are also generated and have been shown to be predictive of measured error rates. Compared to the Applied Biosystems Analysis software, this algorithm generates 18% fewer insertion errors, 80% more deletion errors, and 4% fewer mismatches. The tradeoff between different types of errors can be controlled through a secondary editing step that inserts or deletes base calls depending on their associated confidence values.

Algorithms↗

PubMed2025

Persistent Genomic Erosion in Whooping Cranes Despite Demographic Recovery.

Integrating in-situ (wild) and ex-situ (captive) conservation efforts can mitigate genetic diversity loss and help prevent extinction of endangered wild populations. The whooping crane (Grus americana) experienced severe population declines in the 18th century, culminating in a collapse to ~20 individuals by 1944. Legal protections and conservation actions have since increased the census population from a stock of 16 individuals to approximately 840 individuals, yet the impact on genomic diversity remains unclear. We analysed the temporal dynamics of genomic erosion by sequencing a high-quality reference genome, and re-sequencing 16 historical (years 1867-1893) and 37 modern (2007-2020) genomes, including wild individuals and four generations of captive-bred individuals. Genomic demographic reconstructions reveal a steady decline, accelerating over the past 300 years with the European settlement of North America. Temporal genomic analyses show that despite demographic recovery, the species has lost 70% of its historical genetic diversity and has increased its inbreeding. Although the modern population bottleneck reduced the ancestral genetic load, modern populations possess more realised load than masked load, possibly resulting in a chronic loss of fitness. Integrating pedigree and genomic data, we underscore the role of breeding management in reducing recent inbreeding. Yet ongoing heterozygosity loss, load accumulation, and persistent effects of historical inbreeding (i.e., background inbreeding) argue against the species' downlisting from its current Endangered status on the IUCN Red List and the Endangered Species Act. The presence of private genetic variation in wild and captive populations suggests that wild-captive crosses could enhance genetic diversity and reduce the realised load. Our findings emphasise the role of genomics in informing conservation management and policy.

Animals↗

PubMed1989

Vowel quality changes produced by surrounding tone sequences.

In three experiments, we examined whether energy at the same frequency as one of a vowel's harmonics in the F1 region can be captured by a preceding or following sequence of tones. The position of the /I/-/E/ phoneme boundary along an F1 continuum was used to assess the extent of capture. The first two experiments showed that a sequence of tones at 500 Hz (56-msec duration at 10/sec) can perceptually remove added energy at 500 Hz from a steady vowel (F0 = 125 Hz) that forms part of the sequence. The effect is detectable with one preceding tone, asymptotes with four, and is greater when two tones follow the vowel than when none do. Rising and falling sequences of tones (at 62.5-Hz intervals or at whole-tone intervals) differ in their effect. Falling sequences behave much like constant tones at 500 Hz but with less effect, whereas rising sequences show no evidence of removing the added tone. The second experiment replicated the first and also showed that when the vowel is embedded in a rising or a falling sequence of tones that continue after it, the following tones have no effect. The third experiment suggested that the different effects found with rising versus falling sequences are qualitatively predictable on the basis of the additive effects of their constituent tones rather than by virtue of their contour. The experiments indicated that sequences of repeating tones are much more effective at capturing a harmonic from a vowel than are sequences that follow a simple pattern. This result may reflect the operation of a principle of least commitment in auditory grouping.

Attention↗

PubMedDate not supplied

[Information about the protein secondary structure improves quality of an alignment of protein sequences].

All popular algorithms of pair-wise alignment of protein primary structures (e.g. Smith-Waterman (SW), FASTA, BLAST, et al.) utilize only amino acid sequences. The SW-algorithm is the most accurate among them, i.e. it produces alignments that are most similar to the alignments obtained by superposition of protein 3D-structures. But even the SW-algorithm is unable to restore the 3D-based alignment if similarity of amino acid sequences (%id) is below 30%. We have proposed a novel alignment method that explicitly takes into account the secondary structure of the compared proteins. We have shown that it creates significantly more accurate alignments compared to SW-algorithm. In particular, for sequences with %id < 30% the average accuracy of the new method is 58% compared to 35% for SW-algorithm (the accuracy of an algorithmic sequence alignment is the part of restored position of a "golden standard" alignment obtained by superposition of corresponding 3D-structures). The accuracy of the proposed method is approximately identical both for experimental, and for theoretically predicted secondary structures. Thus the method can be applied for alignment of protein sequences even if protein 3D-structure is unknown. The program is available at ftp://194.149.64.196/STRUSWER/.

Algorithms↗

PubMed1997

Molecular modeling of CD28 and three-dimensional analysis of residue conservation in the CD28/CD152 family.

CD28/CD152-CD80/CD86 receptor-ligand interactions result in costimulatory signals critical for optimal T cell activation. CD28/CD152 and CD80/CD86 are members of the immunoglobulin superfamily (IgSF). Despite common receptor-ligand interactions, both receptor and ligand pairs share only limited sequence identity. A detailed molecular model of the extracellular Ig-like domain of human CD28 was constructed using a combination of different modeling methods. The model was based on the solution structure of CD152 and sequence comparison of the CD28/CD152 family. Assessment of the model revealed good stereochemical quality and sequence-structure compatibility. The CD28 model was used to map surface residues, N-linked glycosylation sites, and to compare residue conservation in CD28 and CD152. The location of N-linked glycosylation sites in CD28/CD152 restricts the surface area available for binding. Rigorous sequence conservation in CD28 and CD152 is limited to core IgSF consensus positions and surface residues implicated in ligand binding. Other surface residues vary greatly in CD28/CD152. Residues critical for ligand binding are surrounded by surface patches conserved only in either CD28 or CD152.

Abatacept↗

PubMed2005

BiQ Analyzer: visualization and quality control for DNA methylation data from bisulfite sequencing.

SUMMARY: Manual processing of DNA methylation data from bisulfite sequencing is a tedious and error-prone task. Here we present an interactive software tool that provides start-to-end support for this process. In an easy-to-use manner, the tool helps the user to import the sequence files from the sequencer, to align them, to exclude or correct critical sequences, to document the experiment, to perform basic statistics and to produce publication-quality diagrams. Emphasis is put on quality control: The program automatically assesses data quality and provides warnings and suggestions for dealing with critical sequences. The BiQ Analyzer program is implemented in the Java programming language and runs on any platform for which a recent Java virtual machine is available. AVAILABILITY: The program is available without charge for non-commercial users and can be downloaded from http://biq-analyzer.bioinf.mpi-inf.mpg.de/

DNA↗

PubMedDate not supplied

Pulse sequence optimization for T2-weighted MR imaging of the brain.

The authors implemented bipolar velocity compensated pulse techniques for T2-weighted MR imaging of the brain. Signal-to-noise (S/N) and image quality was compared for pulse sequences with standard and optimized RF pulses, low and regular bandwidth versions and cardiac triggering. Images from bipolar velocity compensated sequences allowed better visualization of vessels and basilar cisterns and improved image quality relative to standard sequences without velocity compensation. The implementation of optimized RF pulses with bipolar sequences resulted in further improvement in image quality. Single echo sequences consistently had improved image quality and signal-to-noise relative to the second echo of a double echo sequence. Low bandwidth bipolar sequences with extended sampling period had 30% higher S/N, but at the cost of slight loss in edge definition. The highest image quality was obtained with the bipolar, optimized RF, single echo sequence. Using this technique contiguous high quality image slices could be obtained with velocity compensation. The addition of cardiac triggering to bipolar sequences resulted in slight improvement in image quality, but this difference was marginal and probably rarely necessary for MR imaging of the brain.

Brain↗

PubMed2000

Using a neural network for lane-tracking of DNA sequencing slab gels.

High quality lane-tracking of gel images is the first task, and thus a prerequisite, for successful trace processing and base-calling of DNA sequencing slab gels. In most approaches, it is based on statistical calculations, for instance variance and co-variance analysis between neighboring pixel columns in the image. On the basis of these statistical calculations, Kohonen's self-organization neural network model was introduced. We have found that, using several well-structured input data, Kohonen's self-organization neural network model can be trained to fulfill our task of lane-tracking. Furthermore, the quality of lane-tracking could be improved compared to algorithmic approaches.

Algorithms↗

PubMed2006

Robust prescan calibration for multiple spin-echo sequences: application to FSE and b-SSFP.

The collection of fast imaging techniques that use multiple spin-echo (MSE) sequences relies on a precise phase relationship between spin echoes and stimulated echoes that form along the radiofrequency refocusing pulse train. Failure to achieve this condition produces dark banding artifacts that result from destructive interference between signal coherence pathways. Satisfying this condition on the microsecond timescale required is technically challenging for conditions involving strong diffusion-weighted gradients, for arbitrary orientation acquisitions and at large field strengths with high-resolution acquisitions. Two clinically significant MSE sequences, fast spin echo (FSE) and balanced steady-state free precession (b-SSFP), are investigated in this work using a 4-T whole-body scanner. We developed a readout-projection-based prescan technique that ensures coherent signal formation by utilizing banding artifacts to automatically adjust gradient balance. Subsequent image acquisition using the results of this prescan permits the formation of coherent-echo images, which are robust under challenging imaging conditions. The robustness of this approach is demonstrated for FSE and b-SSFP images obtained from the knees of human volunteers. We believe that the use of this prescan calibration technique for the alignment of signal pools in MSE sequences is critical at high fields and will facilitate the implementation of high-quality clinically significant sequences such as FSE and b-SSFP.

Artifacts↗

PubMed1996

The use of an improved transposon mutagenesis system for DNA sequencing leads to the characterization of a new insertion sequence of Streptomyces lividans 66.

A DNA sequencing strategy was developed based on the tetracycline resistance transposon Tn1721. A universal M13 primer binding site (UP) for DNA sequencing and restriction sites for mapping were inserted near one end of Tn1721 and the new derivative, Tn5491, introduced onto a conjugative F' plasmid. The target sequence is inserted between two inverted resolution sites (res) of Tn1721 present on the high-copy plasmid pJOE2114. Due to the inviability of long palindromic sequences in Escherichia coli insertions between the inversely orientated res sites of pJOE2114 are positively selected. Transposition of Tn5491 into the target sequence is selected by cointegrate formation of Tn5491 during transposition, mating and transfer of the nonconjugative sequencing vector. After cointegrate resolution, the additional res sites in the vector result in a second site-specific recombination removing most of the transposon (except of 136 bp) and part of the target sequence. The reduced plasmid sizes and the use of the universal primer improved the quality of the sequencing results obtained on an automated fluorescent sequencer. A 3.35-kb EcoRI fragment from the 30-kb terminal inverted repeats (TIR) of the Streptomyces lividans chromosome was sequenced by this method. A 1304-bp sequence was found on this fragment with the features of insertion elements. The element called IS1372 had 27-bp IR and two potential open reading frames. The predicted gene products had similar sizes and high similarity to gene products encoded by insertion sequences of the IS3 family. Furthermore, a potential signal stimulating ribosomal shifts and typical for members of the IS3 family was identified. Five to seven copies of IS1372 were found in different strains of S. lividans but none in other Streptomyces species tested.

Amino Acid Sequence↗

PubMed1976

Nerve impulse sequences correlated with the four primary taste qualities in rat.

Neural correlates of the taste qualities must be contained in transient impulse trains across many nerve fibres. In the present study, ten chemicals with primary tastes were used in two or three concentrations. Gustatory impulses were recorded from thin strands, consisting of one or at most three functional nerve fibres, dissected out of the chorda tympani nerve of twelve Wistar albino rats, and their numbers in every successive 50 msec were counted for 500 msec period after gustatory stimulus onset. The results obtained were as follows (pless than0.05): 1) Nerve impulse sequence produced by substances of the salty quality were different from those elicited by chemicals of the other three qualities in 250-300 msec after stimulus onset. 2) The same was true in the case of sour and sweet qualities provided that they became dissimilar to each other in 250 msec after stimulation. 3) No difference could be recognized between impulse trains of bitter and sour solutions as well as between those of bitter and sweet ones.

Animals↗

PubMed1992

Rapid and reliable fluorescent cycle sequencing of double-stranded templates.

Automated DNA sequencing is an extremely valuable technique which requires very high quality DNA templates to be carried out successfully. While it has been possible to readily produce large numbers of such templates from M13 or other single-stranded vectors for several years, the sequencing of double-stranded DNA templates using the ABI 373 DNA Sequencer has had a considerably lower success rate. We describe how the combination of a new fluorescent, dideoxy sequencing method, called cycle-sequencing, coupled with modifications to template isolation procedures based on Qiagen columns, makes fluorescent sequencing of double-stranded templates a reliable procedure. From a single five milliliter culture enough DNA can be isolated (up to 20 micrograms) to do 4-8 sequencing reactions, each of which yields 400-500 bases of high quality sequence data. These procedures make the routine use of double-stranded DNA templates a viable strategy in automated DNA sequencing projects.

Base Sequence↗

PubMed2026

Comparison of phylogenetic metrics of transmission between symptomatic and asymptomatic tuberculosis in individuals who were incarcerated in Brazil in 2008-24: a retrospective genomic epidemiology study.

BACKGROUND: Tuberculosis control efforts have traditionally targeted symptomatic individuals; however, the role of asymptomatic cases in sustaining transmission is increasingly recognised. We aimed to quantify the contribution of asymptomatic tuberculosis to recent transmission using genomic and epidemiological data from a high-transmission setting. METHODS: We conducted a retrospective genomic epidemiology study of Mycobacterium tuberculosis isolates collected in Mato Grosso do Sul, Brazil, between Aug 25, 2008, and March 19, 2024. Available isolates underwent whole-genome sequencing. Demographic, clinical, incarceration history, and laboratory metadata were obtained from surveillance records. From Jan 1, 2017, to March 19, 2024, active case finding was conducted in the state's three largest prisons (all male-only facilities), during which sputum samples were collected from individuals irrespective of symptoms and tested using GeneXpert and culture. Comparisons of transmission between individuals with and without symptoms were restricted to individuals who were incarcerated and were identified through active case finding and for whom high-quality, M tuberculosis lineage 4 genomes were available. Metrics of recent transmission included phylogenetic clustering, time-scaled haplotype density (THD), local branching index (LBI), and transmission probabilities inferred using Bayesian Reconstruction and Evolutionary Analysis of Transmission Histories. FINDINGS: 4448 tuberculosis cases were notified in Mato Grosso do Sul in 2008-24. After excluding cases for which M tuberculosis isolates were not available or had low sequencing quality, who had contaminated cultures or mixed infection, or who were infected with non-lineage 4 M tuberculosis, we included 2362 lineage 4 M tuberculosis isolates with high-quality genome sequences. 1849 (78·3%) of 2362 isolates were part of a genomic cluster. Among 2362 individuals with tuberculosis, 1137 (48·1%) were incarcerated at diagnosis. Of these individuals, 505 were identified through active case finding in three male-only prisons. The median age was 30 years (IQR 25-37); 304 (60·2%) had mixed ethnicity, 90 (17·8%) were White, 56 (11·1%) were Black, 13 (2·6%) were Indigenous, and six (1·2%) were Asian. 277 (54·9%) had symptomatic disease and 228 (45·1%) had asymptomatic tuberculosis. There were no significant differences between symptomatic and asymptomatic individuals in phylogenetic clustering (213 [76·9%] of 277 vs 195 [85·5%] of 228; p=0·37), THD (median 0·39 [IQR 0·06-0·62] vs 0·50 [0·09-0·65]; p=0·12), or LBI (0·00863 [0·00810-0·00988] vs 0·00871 [0·00829-0·01020]; p=0·088). Bayesian transmission trees showed no significant difference in the number of secondary infections inferred from symptomatic compared with asymptomatic individuals (p=0·56). These findings were consistent across genomic clusters and robust to model assumptions. INTERPRETATION: We identified no differences in transmission between individuals who were symptomatic and those who were asymptomatic using multiple genomic measures. In this high-transmission setting, where systematic screening is implemented, our findings indicate that asymptomatic tuberculosis substantially contributes to tuberculosis transmission at the population level. These results suggest that symptom-based case detection alone is likely to be insufficient to interrupt transmission and highlight the importance of expanded screening strategies in high-risk populations. FUNDING: US National Institutes of Health and the Brazilian National Research Council (CNPq).

Humans↗

PubMed2004

Quality test of the MicroSeq D2 LSU Fungal Sequencing Kit for the identification of fungi.

Fungal contaminants may be health risk factors in general indoor and occupational environments. The identification of microfungi by macro- and micro-morphological characters is known to be very difficult and time consuming. An alternative method for identification of fungi using a molecular biological method based on a commercially available kit is described. The method is based on the DNA-extraction and subsequent amplification of the D2 expansion region of the large subunit of the fungal rDNA. DNA extraction, amplification, sequencing and database screening can be performed within 24 hours. A total of 28 different fungal species were identified on a morphological basis and compared with identification by the molecular biological method. The database comparison revealed identification down to the species for 12 samples and to the genus level of 11 samples. Five samples could not be identified. It is concluded that the identification of fungi by molecular biological method needs to be improved. This can partly be reached by expanding the fungal DNA-sequence datasets.

Air Pollution, Indoor↗

PubMed1995

A weighting system and algorithm for aligning many phylogenetically related sequences.

Most multiple sequence alignment programs explicitly or implicity try to optimize some score associated with the resulting alignment. Although the sum-of-pairs score is currently most widely used, it is inappropriate when the phylogenetic relationships among the sequences to be aligned are not evenly distributed, since the contributions of densely populated groups dominate those of minor members. This paper proposes an iterative multiple sequence alignment method which optimizes a weighted sum-of-pairs score, in which the weights given to individual sequence pairs are adjusted to compensate for the biased contributions. A simple method that rapidly calculates such a set of weights for a given phylogenetic tree is presented. The multiple sequence alignment is refined through partitioning and realignment restricted to the edges of the tree. Under this restriction, profile-based fast and rigorous group-to-group alignment is achieved at each iteration, rendering the overall computational cost virtually identical to that using an unweighted score. Consistency of nearly 90% was attained between structural and sequence alignments of multiple divergent globins, confirming the effectiveness of this strategy in improving the quality of multiple sequence alignment.

Algorithms↗

PubMed2000

Structural clues in the sequences of the aquaporins.

The large number of sequences available for the aquaporin family represents a valuable source of information to incorporate into three-dimensional structure determination. Phylogenetic analysis was used to define type sequences to avoid extreme over-representation of some subfamilies, and as a measure of the quality of multiple sequence alignment. Inspection of the sequence alignment suggested eight conserved segments that define the core architecture of six transmembrane helices and two functional loops, B and E, projecting into the plane of the membrane. The sum of the core segments and the minimum lengths of the interlinking loops constitute the 208 residues necessary to satisfy the aquaporin architecture. Analysis of hydrophobic and conservation periodicity and of correlated mutations across the alignment indicated the likely assignment and orientation of the helices in the bilayer. This assignment is examined with respect to the structure of the erythrocyte aquaporin 1 determined by electron crystallography. The aquaporin 1 tetramer is described as three rings of helices, each ring with a different exposure to the lipid environment. The sequence analysis clearly suggests that two helices are exposed along their whole lengths, two helices are exposed only at their N termini, and two helices are not exposed to lipid. It is further proposed that, besides loops B and E, the highly conserved motifs on helices 1 and 4, ExxxTxxF/L, could line the water channel.

Amino Acid Sequence↗

PubMed1998

[Ultra-rapid T2-weighted MR imaging during suspended respiration for the examination of focal lesions of the liver: a comparison of TSE, HASTE and HASTE-STIR sequences using a CP body array coil].

PURPOSE: A comparison of ultra rapid T2-weighted HASTE and HASTE-STIR sequences during suspended respiration for the detection of focal lesions of the liver. MATERIAL AND METHODS: Twenty-one patients (59 +/- 12 years) with a total of 33 focal lesions (7 < or = 1 cm, 19 < or = 3 cm; 17 cystic liquid and 16 solid lesions) were examined with a 1.5 T MR apparatus. Sequences: T2 weighted HASTE (TEeff = 64 ms, 90 ms), HASTE-STIR (TEeff = 64 ms, 81 ms) TSE (TE = 132 ms) and T1-weighted FLASH 2D. RESULTS: The T2-weighted TSE and both HASTE sequences showed all the lesions. Two out of the 33 lesions were not demonstrated by the other sequences. Best image quality without movement artefacts and the best resolution of the lesions in the T2-weighted sequences, with the most favourable contrast/noise ratio (36.6 +/- 33.1) and signal/noise ratio (20.8 +/- 10.5) resulted from the HASTE 90, followed by the HASTE 64 sequence (27.5 +/- 24.2; 24.6 +/- 9.1). Both these techniques were superior to the TSE sequence (23.9 +/- 29.4; 13.9 +/- 7.4), which resulted in movement artifacts. With the HASTE-STIR sequences it was possible to show 31 and 32 of the 33 lesions respectively, with very good resolution but poor signal/noise ratio. CONCLUSION: HASTE sequences have the potential of improving the diagnosis of focal liver lesions.

Adenoma↗

PubMed2005

An intein-based genetic selection allows the construction of a high-quality library of binary patterned de novo protein sequences.

Combinatorial libraries of synthetic DNA are increasingly being used to identify and evolve proteins with novel folds and functions. An effective strategy for maximizing the diversity of these libraries relies on the assembly of large genes from smaller fragments of synthetic DNA. To optimize library assembly and screening, it is desirable to remove from the synthetic libraries any sequences that contain unintended frameshifts or stop codons. Although genetic selection systems can be used to accomplish this task, the tendency of individual segments to yield misfolded or aggregated products can decrease the effectiveness of these selections. Furthermore, individual protein domains may misfold when removed from their native context. We report the development and characterization of an in vivo system to preselect sequences that encode uninterrupted gene segments regardless of the foldedness of the encoded polypeptide. In this system, the inserted synthetic gene segment is separated from an intein/thymidylate synthase (TS) reporter domain by a polyasparagine linker, thereby permitting the TS reporter to fold and function independently of the folding and function of the segment-encoded polypeptide. TS-deficient Escherichia coli host cells survive on selective medium only if the insert is uninterrupted and in-frame, thereby allowing selection and amplification of desired sequences. We demonstrate that this system can be used as a highly effective preselection tool for the production of large, diverse and high-quality libraries of de novo protein sequences.

Amino Acid Sequence↗