RESUMEN
KATK is a fast and accurate software tool for calling variants directly from raw next-generation sequencing reads. It uses predefined k-mers to retrieve only the reads of interest from the FASTQ file and calls genotypes by aligning retrieved reads locally. KATK does not use data about known polymorphisms and has NC (no call) as the default genotype. The reference or variant allele is called only if there is sufficient evidence for their presence in data. Thus it is not biased against rare variants or de-novo mutations. With simulated datasets, we achieved a false-negative rate of 0.23% (sensitivity 99.77%) and a false discovery rate of 0.19%. Calling all human exonic regions with KATK requires 1-2 h, depending on sequencing coverage.
Asunto(s)
Análisis Mutacional de ADN/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Programas Informáticos , Algoritmos , Alelos , Mapeo Cromosómico/métodos , Conjuntos de Datos como Asunto , Femenino , Genoma Humano , Genotipo , Humanos , Masculino , Polimorfismo de Nucleótido Simple , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/métodosRESUMEN
Summary: Designing PCR primers for amplifying regions of eukaryotic genomes is a complicated task because the genomes contain a large number of repeat sequences and other regions unsuitable for amplification by PCR. We have developed a novel k-mer based masking method that uses a statistical model to detect and mask failure-prone regions on the DNA template prior to primer design. We implemented the software as a standalone software primer3_masker and integrated it into the primer design program Primer3. Availability and implementation: The standalone version of primer3_masker is implemented in C. The source code is freely available at https://github.com/bioinfo-ut/primer3_masker/ (standalone version for Linux and macOS) and at https://github.com/primer3-org/primer3/ (integrated version). Primer3 web application that allows masking sequences of 196 animal and plant genomes is available at http://primer3.ut.ee/. Contact: maido.remm@ut.ee. Supplementary information: Supplementary data are available at Bioinformatics online.
Asunto(s)
Cartilla de ADN , Reacción en Cadena de la Polimerasa/métodos , Secuencias Repetitivas de Ácidos Nucleicos , Programas Informáticos , Animales , Humanos , Plantas/genéticaRESUMEN
OBJECTIVE: The study aimed to validate a whole-genome sequencing-based NIPT laboratory method and our recently developed NIPTmer aneuploidy detection software with the potential to integrate the pipeline into prenatal clinical care in Estonia. METHOD: In total, 424 maternal blood samples were included. Analysis pipeline involved cell-free DNA extraction, library preparation and massively parallel sequencing on Illumina platform. Aneuploidies were determined with NIPTmer software, which is based on counting pre-defined per-chromosome sets of unique k-mers from sequencing raw data. SeqFF was implemented to estimate cell-free fetal DNA (cffDNA) fraction. RESULTS: NIPTmer identified correctly all samples of non-mosaic trisomy 21 (T21, 15/15), T18 (9/9), T13 (4/4) and monosomy X (4/4) cases, with the 100% sensitivity. However, one mosaic T18 remained undetected. Six false-positive (FP) results were observed (FP rate of 1.5%, 6/398), including three for T18 (specificity 99.3%) and three for T13 (specificity 99.3%). The level of cffDNA of <4% was estimated in eight samples, including one sample with T13 and T18. Despite low cffDNA level, these two samples were determined as aneuploid. CONCLUSION: We believe that the developed NIPT method can successfully be used as a universal primary screening test in combination with ultrasound scan for the first trimester fetal examination.
Asunto(s)
Aneuploidia , Pruebas Prenatales no Invasivas/estadística & datos numéricos , Aberraciones Cromosómicas Sexuales , Programas Informáticos , Estonia , Femenino , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Pruebas Prenatales no Invasivas/métodos , Embarazo , Salud PúblicaRESUMEN
DNA compaction with protamines in sperm is essential for successful fertilization. However, a portion of sperm chromatin remains less tightly packed with histones, which genomic location and function remain unclear. We extracted and sequenced histone-associated DNA from sperm of nine ejaculates from three bulls. We found that the fraction of retained histones varied between samples, but the variance was similar between samples from the same and different individuals. The most conserved regions showed similar abundance across all samples, whereas in other regions, their presence correlated with the size of histone fraction. This may refer to gradual histone-protamine transition, where easily accessible genomic regions, followed by the less accessible regions are first substituted by protamines. Our results confirm those from previous studies that histones remain in repetitive genome elements, such as centromeres, and added new findings of histones in rRNA and SRP RNA gene clusters and indicated histone enrichment in some spermatogenesis-associated genes, but not in genes of early embryonic development. Our functional analysis revealed significant overrepresentation of cGMP-dependent protein kinase G (cGMP-PKG) pathway genes among histone-enriched genes. This pathway is known for its importance in pre-fertilization sperm events. In summary, a novel hypothesis for gradual histone-to-protamine transition in sperm maturation was proposed. We believe that histones may contribute structural information into early embryo by epigenetically modifying centromeric chromatin and other types of repetitive DNA. We also suggest that sperm histones are retained in genes needed for sperm development, maturation and fertilization, as these genes are transcriptionally active shortly prior to histone-to-protamine transition.
Asunto(s)
Cromatina/metabolismo , Epigenómica , Histonas/metabolismo , Protaminas/metabolismo , Espermatogénesis/fisiología , Espermatozoides/metabolismo , Animales , Bovinos , Cromatina/genética , Desarrollo Embrionario/fisiología , Histonas/genética , Masculino , Protaminas/genéticaRESUMEN
Classified into 16 superfamilies, conopeptides are the main component of cone snail venoms that attract growing interest in pharmacology and drug discovery. The conventional approach to assigning a conopeptide to a superfamily is based on a consensus signal peptide of the precursor sequence. While this information is available at the genomic or transcriptomic levels, it is not present in amino acid sequences of mature bioactives generated by proteomic studies. As the number of conopeptide sequences is increasing exponentially with the improvement in sequencing techniques, there is a growing need for automating superfamily elucidation. To face this challenge we have defined distinct models of the signal sequence, propeptide region and mature peptides for each of the superfamilies containing more than 5 members (14 out of 16). These models rely on two robust techniques namely, Position-Specific Scoring Matrices (PSSM, also named generalized profiles) and hidden Markov models (HMM). A total of 50 PSSMs and 47 HMM profiles were generated. We confirm that propeptide and mature regions can be used to efficiently classify conopeptides lacking a signal sequence. Furthermore, the combination of all three-region models demonstrated improvement in the classification rates and results emphasise how PSSM and HMM approaches complement each other for superfamily determination. The 97 models were validated and offer a straightforward method applicable to large sequence datasets.
Asunto(s)
Aminoácidos , Caracol Conus , Péptidos , Análisis de Secuencia de Proteína , Aminoácidos/genética , Aminoácidos/metabolismo , Animales , Biología Computacional , Caracol Conus/química , Caracol Conus/genética , Cadenas de Markov , Péptidos/clasificación , Péptidos/genética , Péptidos/metabolismo , Ponzoñas/químicaRESUMEN
ConoDictor is a tool that enables fast and accurate classification of conopeptides into superfamilies based on their amino acid sequence. ConoDictor combines predictions from two complementary approaches-profile hidden Markov models and generalized profiles. Results appear in a browser as tables that can be downloaded in various formats. This application is particularly valuable in view of the exponentially increasing number of conopeptides that are being identified. ConoDictor was written in Perl using the common gateway interface module with a php submission page. Sequence matching is performed with hmmsearch from HMMER 3 and ps_scan.pl from the pftools 2.3 package. ConoDictor is freely accessible at http://conco.ebc.ee.
Asunto(s)
Conotoxinas/clasificación , Programas Informáticos , Conotoxinas/química , Internet , Cadenas de Markov , Análisis de Secuencia de Proteína , Interfaz Usuario-ComputadorRESUMEN
Conopeptides are small toxins produced by predatory marine snails of the genus Conus. They are studied with increasing intensity due to their potential in neurosciences and pharmacology. The number of existing conopeptides is estimated to be 1 million, but only about 1000 have been described to date. Thanks to new high-throughput sequencing technologies the number of known conopeptides is likely to increase exponentially in the near future. There is therefore a need for a fast and accurate computational method for identification and classification of the novel conopeptides in large data sets. 62 profile Hidden Markov Models (pHMMs) were built for prediction and classification of all described conopeptide superfamilies and families, based on the different parts of the corresponding protein sequences. These models showed very high specificity in detection of new peptides. 56 out of 62 models do not give a single false positive in a test with the entire UniProtKB/Swiss-Prot protein sequence database. Our study demonstrates the usefulness of mature peptide models for automatic classification with accuracy of 96% for the mature peptide models and 100% for the pro- and signal peptide models. Our conopeptide profile HMMs can be used for finding and annotation of new conopeptides from large datasets generated by transcriptome or genome sequencing. To our knowledge this is the first time this kind of computational method has been applied to predict all known conopeptide superfamilies and some conopeptide families.
Asunto(s)
Conotoxinas/clasificación , Caracol Conus/química , Neurotoxinas/clasificación , Precursores de Proteínas/clasificación , Transcriptoma , Secuencia de Aminoácidos , Animales , Conotoxinas/química , Conotoxinas/aislamiento & purificación , Caracol Conus/genética , Bases de Datos de Proteínas , Cadenas de Markov , Datos de Secuencia Molecular , Neurotoxinas/química , Neurotoxinas/aislamiento & purificación , Filogenia , Precursores de Proteínas/química , Precursores de Proteínas/aislamiento & purificación , Señales de Clasificación de Proteína/fisiología , Análisis de Secuencia de Proteína , Terminología como AsuntoRESUMEN
Motivation: Accurate estimation of next-generation sequencing depth of coverage is needed for detecting the copy number of repeated elements in the human genome. The common methods for estimating sequencing depth are based on counting the number of reads mapped to the genome or subgenomic regions. Such methods are sensitive to the mapping quality. The presence of contamination or the large deviance of an individual genome from the reference may introduce bias in depth estimation. Results: Here, we present an algorithm and implementation for estimating both the sequencing depth and error rate from unmapped reads using a uniquely filtered k-mer set. On simulated reads with 20× coverage, the margin of error was less than 0.01%. At 0.01× coverage and the presence of 10-fold contamination, the precision was within 2% for depth and within 10% for error rate. Availability and implementation: DOCEST program and database can be downloaded from https://bioinfo.ut.ee/docest/. Supplementary information: Supplementary data are available at Bioinformatics Advances online.
RESUMEN
BACKGROUND: We present a comprehensive technological solution for bacterial diagnostics using tmRNA as a marker molecule. A robust probe design algorithm for microbial detection microarray is implemented. The probes were evaluated for specificity and, combined with NASBA (Nucleic Acid Sequence Based Amplification) amplification, for sensitivity. RESULTS: We developed a new web-based program SLICSel for the design of hybridization probes, based on nearest-neighbor thermodynamic modeling. A SLICSel minimum binding energy difference criterion of 4 kcal/mol was sufficient to design of Streptococcus pneumoniae tmRNA specific microarray probes. With lower binding energy difference criteria, additional hybridization specificity tests on the microarray were needed to eliminate non-specific probes. Using SLICSel designed microarray probes and NASBA we were able to detect S. pneumoniae tmRNA from a series of total RNA dilutions equivalent to the RNA content of 0.1-10 CFU. CONCLUSIONS: The described technological solution and both its separate components SLICSel and NASBA-microarray technology independently are applicative for many different areas of microbial diagnostics.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Bacteriano/genética , Replicación de Secuencia Autosostenida/métodos , Streptococcus pneumoniae/genética , Sondas ARN/genética , Programas Informáticos , Especificidad de la EspecieRESUMEN
BACKGROUND: The hybridization of synthetic Streptococcus pneumoniae tmRNA on a detection microarray is slow at 34 degrees C resulting in low signal intensities. RESULTS: We demonstrate that adding specific DNA helper oligonucleotides (chaperones) to the hybridization buffer increases the signal strength at a given temperature and thus makes the specific detection of Streptococcus pneumoniae tmRNA more sensitive. No loss of specificity was observed at low temperatures compared to hybridization at 46 degrees C. The effect of the chaperones can be explained by disruption of the strong secondary and tertiary structure of the target RNA by the selective hybridization of helper molecules. The amplification of the hybridization signal strength by chaperones is not necessarily local; we observed increased signal intensities in both local and distant regions of the target molecule. CONCLUSIONS: The sensitivity of the detection of tmRNA at low temperature can be increased by chaperone oligonucleotides. Due to the complexity of RNA secondary and tertiary structures the effect of any individual chaperone is currently not predictable.
Asunto(s)
Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , Oligonucleótidos/química , ARN Bacteriano/aislamiento & purificación , Secuencia de Bases , Datos de Secuencia Molecular , Conformación de Ácido Nucleico , Sensibilidad y Especificidad , Streptococcus pneumoniae/genética , TemperaturaRESUMEN
BACKGROUND: Here we present a novel promising microbial diagnostic method that combines the sensitivity of Nucleic Acid Sequence Based Amplification (NASBA) with the high information content of microarray technology for the detection of bacterial tmRNA molecules. The NASBA protocol was modified to include aminoallyl-UTP (aaUTP) molecules that were incorporated into nascent RNA during the NASBA reaction. Post-amplification labeling with fluorescent dye was carried out subsequently and tmRNA hybridization signal intensities were measured using microarray technology. Significant optimization of the labeled NASBA protocol was required to maintain the required sensitivity of the reactions. RESULTS: Two different aaUTP salts were evaluated and optimum final concentrations were identified for both. The final 2 mM concentration of aaUTP Li-salt in NASBA reaction resulted in highest microarray signals overall, being twice as high as the strongest signals with 1 mM aaUTP Na-salt. CONCLUSION: We have successfully demonstrated efficient combination of NASBA amplification technology with microarray based hybridization detection. The method is applicative for many different areas of microbial diagnostics including environmental monitoring, bio threat detection, industrial process monitoring and clinical microbiology.
Asunto(s)
Colorantes Fluorescentes/química , Análisis de Secuencia por Matrices de Oligonucleótidos/métodos , ARN Bacteriano/química , Replicación de Secuencia Autosostenida/métodos , Sensibilidad y Especificidad , Streptococcus pneumoniae/metabolismo , Uridina Trifosfato/químicaRESUMEN
Non-invasive prenatal testing (NIPT) is a recent and rapidly evolving method for detecting genetic lesions, such as aneuploidies, of a fetus. However, there is a need for faster and cheaper laboratory and analysis methods to make NIPT more widely accessible. We have developed a novel software package for detection of fetal aneuploidies from next-generation low-coverage whole genome sequencing data. Our tool - NIPTmer - is based on counting pre-defined per-chromosome sets of unique k-mers from raw sequencing data, and applying linear regression model on the counts. Additionally, the filtering process used for k-mer list creation allows one to take into account the genetic variance in a specific sample, thus reducing the source of uncertainty. The processing time of one sample is less than 10 CPU-minutes on a high-end workstation. NIPTmer was validated on a cohort of 583 NIPT samples and it correctly predicted 37 non-mosaic fetal aneuploidies. NIPTmer has the potential to reduce significantly the time and complexity of NIPT post-sequencing analysis compared to mapping-based methods. For non-commercial users the software package is freely available at http://bioinfo.ut.ee/NIPTMer/ .
Asunto(s)
Aneuploidia , Feto/metabolismo , Pruebas Genéticas/métodos , Interfaz Usuario-Computador , Adulto , Ácidos Nucleicos Libres de Células/química , Ácidos Nucleicos Libres de Células/aislamiento & purificación , Femenino , Variación Genética , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Embarazo , Atención Prenatal , Análisis de Secuencia de ADNRESUMEN
In this chapter, we describe MultiPLX-a tool for automatic grouping of PCR primers for multiplexed PCR. Both generic working principle and step-by-step practical procedures with examples are presented. MultiPLX performs grouping by calculating many important interaction levels between the different primer pairs and then distributes primer pairs to groups, so that the strength of unwanted interactions is kept below user-defined compatibility level. In addition, it can be used to select optimal primer pairs for multiplexing from list of candidates. MultiPLX can be downloaded from http://bioinfo.ut.ee/download/. Graphical Web-based interface to most functions of MultiPLX is available at http://bioinfo.ut.ee/multiplx/.
Asunto(s)
Cartilla de ADN/química , Internet , Reacción en Cadena de la Polimerasa , Análisis de Secuencia de ADN , Programas Informáticos , Interfaz Usuario-Computador , Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Análisis de Secuencia de ADN/métodosRESUMEN
In this article, we describe the working principle and a list of practical applications for GenomeMasker-a program that finds and masks all repeated DNA motifs in fully sequenced genomes. The GenomeMasker exhaustively finds and masks all repeated DNA motifs in studied genomes. The software is optimized for polymerase chain reaction (PCR) primer design. The algorithm is designed for high-throughput work, allowing masking of large DNA regions, even entire eukaryotic genomes. Additionally, the software is able to predict all alternative PCR products from studied genomes for thousands of candidate PCR primer pairs. Practical applications of the GenomeMasker are shown for command-line version of the GenomeMasker, which can be downloaded from http://bioinfo.ut.ee/download/. Graphical Web interfaces with limited options are available at http://bioinfo.ut.ee/genometester/ and http://bioinfo.ut.ee/snpmasker/.
Asunto(s)
Células Eucariotas , Genoma , Reacción en Cadena de la Polimerasa , Secuencias Repetitivas de Ácidos Nucleicos , Análisis de Secuencia de ADN , Programas Informáticos , Animales , Humanos , Internet , Reacción en Cadena de la Polimerasa/métodos , Valor Predictivo de las Pruebas , Análisis de Secuencia de ADN/métodosRESUMEN
We have developed a computational method that counts the frequencies of unique k-mers in FASTQ-formatted genome data and uses this information to infer the genotypes of known variants. FastGT can detect the variants in a 30x genome in less than 1 hour using ordinary low-cost server hardware. The overall concordance with the genotypes of two Illumina "Platinum" genomes is 99.96%, and the concordance with the genotypes of the Illumina HumanOmniExpress is 99.82%. Our method provides k-mer database that can be used for the simultaneous genotyping of approximately 30 million single nucleotide variants (SNVs), including >23,000 SNVs from Y chromosome. The source code of FastGT software is available at GitHub (https://github.com/bioinfo-ut/GenomeTester4/).
Asunto(s)
Algoritmos , Genoma Humano , Polimorfismo de Nucleótido Simple , Análisis de Secuencia de ADN/métodos , Programas Informáticos , Teorema de Bayes , Benchmarking , Genotipo , Secuenciación de Nucleótidos de Alto Rendimiento , Humanos , Reproducibilidad de los Resultados , Análisis de Secuencia de ADN/estadística & datos numéricosRESUMEN
BACKGROUND: Fast, accurate and high-throughput identification of bacterial isolates is in great demand. The present work was conducted to investigate the possibility of identifying isolates from unassembled next-generation sequencing reads using custom-made guide trees. RESULTS: A tool named StrainSeeker was developed that constructs a list of specific k-mers for each node of any given Newick-format tree and enables the identification of bacterial isolates in 1-2 min. It uses a novel algorithm, which analyses the observed and expected fractions of node-specific k-mers to test the presence of each node in the sample. This allows StrainSeeker to determine where the isolate branches off the guide tree and assign it to a clade whereas other tools assign each read to a reference genome. Using a dataset of 100 Escherichia coli isolates, we demonstrate that StrainSeeker can predict the clades of E. coli with 92% accuracy and correct tree branch assignment with 98% accuracy. Twenty-five thousand Illumina HiSeq reads are sufficient for identification of the strain. CONCLUSION: StrainSeeker is a software program that identifies bacterial isolates by assigning them to nodes or leaves of a custom-made guide tree. StrainSeeker's web interface and pre-computed guide trees are available at http://bioinfo.ut.ee/strainseeker. Source code is stored at GitHub: https://github.com/bioinfo-ut/StrainSeeker.
RESUMEN
BACKGROUND: The design of oligonucleotides and PCR primers for studying large genomes is complicated by the redundancy of sequences. The eukaryotic genomes are particularly difficult to study due to abundant repeats. The speed of most existing primer evaluation programs is not sufficient for large-scale experiments. RESULTS: In order to improve the efficiency and success rate of automatic primer/oligo design, we created a novel method which allows rapid masking of repeats in large sequence files, for example in eukaryotic genomes. It also allows the detection of all alternative binding sites of PCR primers and the prediction of PCR products. The new method was implemented in a collection of efficient programs, the GENOMEMASKER package. The performance of the programs was compared to other similar programs. We also modified the PRIMER3 program, to be able to design primers from lowercase-masked sequences. CONCLUSION: The GENOMEMASKER package is able to mask the entire human genome for non-unique primers within 6 hours and find locations of all binding sites for 10,000 designed primer pairs within 10 minutes. Additionally, it predicts all alternative PCR products from large genomes for given primer pairs.
Asunto(s)
Cartilla de ADN/genética , Genoma Humano , Reacción en Cadena de la Polimerasa/métodos , Secuencia de Bases/genética , Sitios de Unión/genética , Humanos , Datos de Secuencia MolecularRESUMEN
In this chapter we describe MultiPLX-a tool for automatic grouping of PCR primers for multiplexed PCR. Both generic working principle and step-by-step practical procedures with examples are presented. MultiPLX performs grouping by calculating many important interaction levels between the different primer pairs and then distributes primer pairs to groups so that the strength of unwanted interactions is kept below user-defined compatibility level. In addition it can be used to select optimal primer pairs for multiplexing from list of candidates. MultiPLX can be downloaded from http://bioinfo.ut.ee/?page_id=167. Graphical web-based interface to most functions of MultiPLX is available at http://bioinfo.ut.ee/multiplx/.
Asunto(s)
Cartilla de ADN/genética , Reacción en Cadena de la Polimerasa/métodos , Animales , HumanosRESUMEN
In this article we describe the working principle and a list of practical applications for GenomeMasker-a program that finds and masks all repeated DNA motifs in fully sequenced genomes. The GenomeMasker exhaustively finds and masks all repeated DNA motifs in studied genomes. The software is optimized for PCR primer design. The algorithm is designed for high-throughput work, allowing masking of large DNA regions, even entire eukaryotic genomes. Additionally, the software is able to predict all alternative PCR products from studied genomes for thousands of candidate PCR primer pairs. Practical applications of the GenomeMasker are shown for command-line version of the GenomeMasker, which can be downloaded from http://bioinfo.ut.ee/download/. Graphical Web interfaces with limited options are available at http://bioinfo.ut.ee/genometester/ and http://bioinfo.ut.ee/snpmasker/.
Asunto(s)
Cartilla de ADN/metabolismo , Células Eucariotas/metabolismo , Genoma/genética , Algoritmos , Sitios de Unión/genética , Programas InformáticosRESUMEN
BACKGROUND: K-mer-based methods of genome analysis have attracted great interest because they do not require genome assembly and can be performed directly on sequencing reads. Many analysis tasks require one to compare k-mer lists from different sequences to find words that are either unique to a specific sequence or common to many sequences. However, no stand-alone k-mer analysis tool currently allows one to perform these algebraic set operations. FINDINGS: We have developed the GenomeTester4 toolkit, which contains a novel tool GListCompare for performing union, intersection and complement (difference) set operations on k-mer lists. We provide examples of how these general operations can be combined to solve a variety of biological analysis tasks. CONCLUSIONS: GenomeTester4 can be used to simplify k-mer list manipulation for many biological analysis tasks.