Triplet Periodicity Research Articles

Search for Dispersed Repeats in Bacterial Genomes Using an Iterative Procedure

We have developed a de novo method for the identification of dispersed repeats based on the use of random position-weight matrices (PWMs) and an iterative procedure (IP). The created algorithm (IP method) allows detection of dispersed repeats for which the average number of substitutions between any two repeats per nucleotide (x) is less than or equal to 1.5. We have shown that all previously developed methods and algorithms (RED, RECON, and some others) can only find dispersed repeats for x ≤ 1.0. We applied the IP method to find dispersed repeats in the genomes of E. coli and nine other bacterial species. We identify three families of approximately 1.09 × 106, 0.64 × 106, and 0.58 × 106 DNA bases, respectively, constituting almost 50% of the complete E. coli genome. The length of the repeats is in the range of 400 to 600 bp. Other analyzed bacterial genomes contain one to three families of dispersed repeats with a total number of 103 to 6 × 103 copies. The existence of such highly divergent repeats could be associated with the presence of a single-type triplet periodicity in various genes or with the packing of bacterial DNA into a nucleoid.

Beginners guide to ribosome profiling

Beginners guide to ribosome profiling

Small-scale sequencing enables quality assessment of Ribo-Seq data: an example from Arabidopsis cell culture

BackgroundTranslation is a tightly regulated process, controlling the rate of protein synthesis in cells. Ribosome sequencing (Ribo-Seq) is a recently developed tool for studying actively translated mRNA and can thus directly address translational regulation. Ribo-Seq libraries need to be sequenced to a great depth due to high contamination by rRNA and other contaminating nucleic acid fragments. Deep sequencing is expensive, and it generates large volumes of data, making data analysis complicated and time consuming.Methods and resultsHere we developed a platform for Ribo-Seq library construction and data analysis to enable rapid quality assessment of Ribo-Seq libraries with the help of a small-scale sequencer. Our data show that several qualitative features of a Ribo-Seq library, such as read length distribution, P-site distribution, reading frame and triplet periodicity, can be effectively evaluated using only the data generated by a benchtop sequencer with a very limited number of reads.ConclusionOur pipeline enables rapid evaluation of Ribo-Seq libraries, opening up possibilities for optimization of Ribo-Seq library construction from difficult samples, and leading to better decision making prior to more costly deep sequencing.

Coding Structure for the ORF1ab, S, M and N Coronavirus Genes

Spectral-statistical approach was applied to comparative analysis of coronavirus genomes from the four genus Alphacoronavirus, Betacoronavirus (including new SARS-CoV-2 virus), Gammacoronavirus and Deltacoronavirus. This analysis was done from the point of view of 3-regularity and latent triplet profile periodicity existence in the coding sequences of four structural genes: ORF1ab encoding transcriptase; S-gene of glycoprotein forming spikes; M-gene of membrane protein; N-gene of nucleoprotein. A whole number of the genomes analyzed was equal to 3410. Gene numbers in each of the four groups in the study respectively were the same. In the result, practically, in the CDSs of all analyzed genes of ORF1ab, S and N the latent profile triplet periodicity was revealed and high value of 3-regularity index, being a quality estimate of coding triplet structure conservation, was determined. On the contrary, for coding structure of M-genes a tendency was revealed to diffuse up to homogeneity for 60 % of the genes in the genomes of alphacoronaviruses analyzed and for 67 % of the genes of the gammacoronaviruses. Tendency of the such structure diffusion, being accompanied by decrease of 3-regularity index average value in comparison with other genes, while the triplet profile periodicity remains saved, was also noted for M-genes of SARS-CoV-2 viruses. Probably, this tendency reflects a significance of M-genes variability in coronavirus adaptation to the novel hosts of genus. Analysis of 3-profile periodicity matrices of the four groups of SARS-CoV-2 genes considered in the work, for the viruses isolated in Europe, Asia and USA, did not revealed their significant difference, that is allowing to propose a single source of this virus propagation.

A Spectral Rotation Method with Triplet Periodicity Property for Planted Motif Finding Problems.

Genes are known as functional patterns in the genome and are presumed to have biological significance. They can indicate binding sites for transcription factors and they encode certain proteins. Finding genes from biological sequences is a major task in computational biology for unraveling the mechanisms of gene expression. Planted motif finding problems are a class of mathematical models abstracted from the process of detecting genes from genome, in which a specific gene with a number of mutations is planted into a randomly generated background sequence, and then gene finding algorithms can be tested to check if the planted gene can be found in feasible time. In this work, a spectral rotation method based on triplet periodicity property is proposed to solve planted motif finding problems. The proposed method gives significant tolerance of base mutations in genes. Specifically, genes having a number of substitutions can be detected from randomly generated background sequences. Experimental results on genomic data set from Saccharomyces cerevisiae reveal that genes can be visually distinguished. It is proposed that genes with about 50% mutations can be detected from randomly generated background sequences. It is found that with about 5 insertions or deletions, this method fails in finding the planted genes. For a particular case, if the deletion of bases is located at the beginning of the gene, that is, bases are not randomly deleted, then the tolerance of the method for base deletion is increased.

ПРОГРАМНЕ ЗАБЕЗПЕЧЕННЯ ДЛЯ ПОШУКУ ТОЧОК ЗМІНИ ТРИПЛЕТНОЇ ПЕРІОДИЧНОСТІ

With the advent of new generation sequencing methods, an exponential increase in molecular-biological data is observed. Data banks of biological sequences, such as GenBank [1], UniProt [2], KEGG [3] and others have reached large sizes. At the same time, the velocity of the description of the sequenced sequences is considerably behind the rate of their accumulation. With such an increase in the volume of information, solving the problem of lagging is impossible without the use of effective algorithms. A rather interesting, complex and deserving attention and detailed study is the direction of genomics responsible for finding efficient algorithms for solving the problem of pro and eukaryote gene identification. The most popular are software products that allow finding genes in accordance with the received pairwise alignment of the sequences using the dynamic programming method. This approach does not allow to explore all areas of the nucleotide sequence, since most of the genes have evolved during evolution, for example, by point mutations or the encoding region of the gene was obtained due to the incorporation of several genes. In such a situation, information about the sites forming hybrid genes will not be available in the databases. Noteworthy is the non-trivial algorithm that was described in [4]. Finding the points of triplet periodicity change for a direct and backward complementary sequence enables the definition of coordinates that may indicate the location of the combination of the two genes.

Deficiency of mitoribosomal S10 protein affects translation and splicing in Arabidopsis mitochondria.

The ribosome is not only a protein-making machine, but also a regulatory element in protein synthesis. This view is supported by our earlier data showing that Arabidopsis mitoribosomes altered due to the silencing of the nuclear RPS10 gene encoding mitochondrial ribosomal protein S10 differentially translate mitochondrial transcripts compared with the wild-type. Here, we used ribosome profiling to determine the contribution of transcriptional and translational control in the regulation of protein synthesis in rps10 mitochondria compared with the wild-type ones. Oxidative phosphorylation system proteins are preferentially synthesized in wild-type mitochondria but this feature is lost in the mutant. The rps10 mitoribosomes show slightly reduced translation efficiency of most respiration-related proteins and at the same time markedly more efficiently synthesize ribosomal proteins and MatR and TatC proteins. The mitoribosomes deficient in S10 protein protect shorter transcript fragments which exhibit a weaker 3-nt periodicity compared with the wild-type. The decrease in the triplet periodicity is particularly drastic for genes containing introns. Notably, splicing is considerably less effective in the mutant, indicating an unexpected link between the deficiency of S10 and mitochondrial splicing. Thus, a shortage of the mitoribosomal S10 protein has wide-ranging consequences on mitochondrial gene expression.

New Method for Potential Fusions Detection in Protein-Coding Sequences.

Gene fusion is known to be one of the mechanisms of a new gene formation. Most bioinformatics methods for studying fused genes are based on the sequence similarity search. However, if the ancestral sequences were lost during evolution or changed too much, it is impossible to detect the fusion. Previously, we have developed a method of searching for triplet periodicity (TP) change points in protein-coding sequences (CDS) and showed the possible relation of this phenomenon with gene formation as a result of fusion. In this study, we improved the TP change point detection method and studied the genes of six eukaryotic genomes. At the level of 2%-3% of the probability of type I error, TP change points were found in 20%-40% of genes. Further analysis showed that about 30% of the TP change points can be explained by amino acid repeats. Another 30% can be potentially fused genes, alignment for which was detected by the BLAST program. We believe that the rest of the results can be fused genes, the ancestral sequences for which have been lost. The method is more sensitive to TP changes and allowed us to find up to two to three times more cases of significant TP change points than our previous method.

Prediction of Sphingosine protein-coding regions with a self adaptive spectral rotation method.

Identifying protein coding regions in DNA sequences by computational methods is an active research topic. Welan gum produced by Sphingomonas sp. WG has great application potential in oil recovery and concrete construction industry. Predicting the coding regions in the Sphingomonas sp. WG genome and addressing the mechanism underlying the explanation for the synthesis of Welan gum metabolism is an important issue at present. In this study, we apply a self adaptive spectral rotation (SASR, for short) method, which is based on the investigation of the Triplet Periodicity property, to predict the coding regions of the whole-genome data of Sphingomonas sp. WG without any previous training process, and 1115 suspected gene fragments are obtained. Suspected gene fragments are subjected to a similarity search against the non-redundant protein sequences (nr) database of NCBI with blastx, and 762 suspected gene fragments have been labeled as genes in the nr database.

Search for potential reading frameshifts in cds from Arabidopsis thaliana and other genomes.

A new mathematical method for potential reading frameshift detection in protein-coding sequences (cds) was developed. The algorithm is adjusted to the triplet periodicity of each analysed sequence using dynamic programming and a genetic algorithm. This does not require any preliminary training. Using the developed method, cds from the Arabidopsis thaliana genome were analysed. In total, the algorithm found 9,930 sequences containing one or more potential reading frameshift(s). This is ∼21% of all analysed sequences of the genome. The Type I and Type II error rates were estimated as 11% and 30%, respectively. Similar results were obtained for the genomes of Caenorhabditis elegans, Drosophila melanogaster, Homo sapiens, Rattus norvegicus and Xenopus tropicalis. Also, the developed algorithm was tested on 17 bacterial genomes. We compared our results with the previously obtained data on the search for potential reading frameshifts in these genomes. This study discussed the possibility that the reading frameshift seems like a relatively frequently encountered mutation; and this mutation could participate in the creation of new genes and proteins.

RPS15 mutations Repress mRNA Translation in Chronic Lymphocytic Leukemia Cells

We and others recently reported mutations within the RPS15 gene, encoding a component of the 40S ribosomal subunit, in clinically aggressive chronic lymphocytic leukemia (CLL). RPS15 mutations resided within an evolutionary conserved region, alluding to an oncogenic rather than a tumor-suppressor role. Our pilot functional analysis revealed that, similar to other ribosomal proteins (RPs), RPS15 also binds MDM2 and may impact on the p53 response. Here, we performed ribosome profiling in order to gain global insight into changes in translation induced by RPS15 mutations in CLL cells. This technique involves measuring translational efficiency (TE), by comparing the levels of ribosome-associated mRNA footprints against the total mRNA for each gene. For 6 CLL cases bearing mutant (mut, n=3) or wildtype (wt, n=3) RPS15, we obtained both ribosome-protected footprints (RPFs) and matching mRNA sequencing data. In parallel, we created stable MEC1 CLL cell lines expressing an additional copy of wt or mut RPS15 (131S) by lentiviral transduction; validation of the transgene expression was performed by Sanger sequencing of amplified cDNAs. Ribosome footprinting and subsequent library preparation of RPFs and total mRNA for all samples was performed with the Illumina Truseq Ribo Profile Kit and all libraries were sequenced on a NextSeq500 instrument. Reads were aligned to the human hg19 genome using Bowtie2. SystemPipeR was used to determine the percentage of reads mapping to 5‘ UTRs, CDS, and 3‘ UTRs and triplet periodicity was assessed using RibORF. The RPFs were of high quality, as assessed by expected RPF size (28-30nt), CDS enrichment, and triplet periodicity.To determine differentially expressed genes between RPS15-mut vs RPS15-wt cases we used DESeq2 while, for differentially translated genes we used Xtail. Changes in transcription and translation between PRS15-wt vs RPS15-mut cases showed limited overlap in both primary CLL cells and cell lines (12.8% and 12.9%, respectively), indicating the potential of ribosome profiling to reveal additional information compared to RNA sequencing alone. In primary CLL cells, 474 genes showed differences only at the transcription level (log2FC mRNA>I1I, p<0.05), while 742 genes were modulated only at the translation level (log2FC RPF>I1I, p<0.05). We identified 322 genes with differential TE (log2FC TE<I1I, p<0.05) between PRS15-wt vs RPS15-mut CLL cases; 262/322 (81%) showed reduced TE in RPS15-mut versus RPS15-wt cases. Similar analysis for the stable MEC1 cell lines revealed 749 genes displaying differences only at the transcription level (log2FC mRNA>I1I), while 1859 genes were regulated only at the translation level (log2FC RPF>I1I). Overall, 771 genes displayed differential TE (log2FC TE<I1I, adjusted p<0.1) between PRS15-wt vs RPS15-mut MEC1 cell lines; 48% of the genes showed reduced TE in mut vs wt cell lines and the remaining 52% augmented TE. The slightly different results compared to those obtained from primary CLL cells, may be attributed to the following reasons: (i) MEC1 cells are TP53-aberrant; (ii) the PRS15-wt cell line overexpresses the RPS15 gene compared to primary CLL cells; and,( iii) the RPS15-mut cell line expresses both the wt and mut RPS15 mRNAs (22% of the mapped reads correspond to the mut RPS15 and 78% to the wt gene, respectively). Gene ontology analysis (Enrichr) of the genes showing differential TE, revealed that in both primary CLL cells and MEC1 cell lines a large fraction of the deregulated transcripts is implicated in RNA binding processes (adj-p=0.0001; adj-p=1.98X10^-13, respectively) which are known to induce translational repression. Interestingly, in primary CLL cells, amongst genes with reduced TE we identified genes implicated in tRNA biosynthesis, protein processing in the endoplasmatic reticulum and the Hippo signaling pathway (p<0.01). Additionally, enrichment analysis revealed that a proportion of genes with reduced TE were targets of the MYC transcription factor (adj-p=0.0005). RP genes, despite unchanged mRNA levels, showed changes in RPF levels and differential TE, suggesting that RPs are also deregulated at the translational level. In conclusion, we show that RPS15 mutations rewire the translation program of CLL cells by reducing the TE of critical molecules, including translation initiation factors and other regulatory elements. DisclosuresHadzidimitriou:Abbvie: Research Funding; Gilead: Research Funding; Janssen: Honoraria, Research Funding. Stamatopoulos:Janssen: Honoraria, Research Funding; Abbvie: Honoraria, Research Funding; Gilead: Honoraria, Research Funding.

The exon-intron gene structure upstream of the initiation codon predicts translation efficiency.

Introns in mRNA leaders are common in complex eukaryotes, but often overlooked. These introns are spliced out before translation, leaving exon-exon junctions in the mRNA leaders (leader EEJs). Our multi-omic approach shows that the number of leader EEJs inversely correlates with the main protein translation, as does the number of upstream open reading frames (uORFs). Across the five species studied, the lowest levels of translation were observed for mRNAs with both leader EEJs and uORFs (29%). This class of mRNAs also have ribosome footprints on uORFs, with strong triplet periodicity indicating uORF translation. Furthermore, the positions of both leader EEJ and uORF are conserved between human and mouse. Thus, the uORF, in combination with leader EEJ predicts lower expression for nearly one-third of eukaryotic proteins.

GWIPS-viz: 2018 update.

The GWIPS-viz browser (http://gwips.ucc.ie/) is an on-line genome browser which is tailored for exploring ribosome profiling (Ribo-seq) data. Since its publication in 2014, GWIPS-viz provides Ribo-seq data for an additional 14 genomes bringing the current total to 23. The integration of new Ribo-seq data has been automated thereby increasing the number of available tracks to 1792, a 10-fold increase in the last three years. The increase is particularly substantial for data derived from human sources. Following user requests, we added the functionality to download these tracks in bigWig format. We also incorporated new types of data (e.g. TCP-seq) as well as auxiliary tracks from other sources that help with the interpretation of Ribo-seq data. Improvements in the visualization of the data have been carried out particularly for bacterial genomes where the Ribo-seq data are now shown in a strand specific manner. For higher eukaryotic datasets, we provide characteristics of individual datasets using the RUST program which includes the triplet periodicity, sequencing biases and relative inferred A-site dwell times. This information can be used for assessing the quality of Ribo-seq datasets. To improve the power of the signal, we aggregate Ribo-seq data from several studies into Global aggregate tracks for each genome.

RiboGalaxy: A browser based platform for the alignment, analysis and visualization of ribosome profiling data

ABSTRACTRibosome profiling (ribo-seq) is a technique that uses high-throughput sequencing to reveal the exact locations and densities of translating ribosomes at the entire transcriptome level. The technique has become very popular since its inception in 2009. Yet experimentalists who generate ribo-seq data often have to rely on bioinformaticians to process and analyze their data. We present RiboGalaxy (http://ribogalaxy.ucc.ie), a freely available Galaxy-based web server for processing and analyzing ribosome profiling data with the visualization functionality provided by GWIPS-viz (http://gwips.ucc.ie). RiboGalaxy offers researchers a suite of tools specifically tailored for processing ribo-seq and corresponding mRNA-seq data. Researchers can take advantage of the published workflows which reduce the multi-step alignment process to a minimum of inputs from the user. Users can then explore their own aligned data as custom tracks in GWIPS-viz and compare their ribosome profiles to existing ribo-seq tracks from published studies. In addition, users can assess the quality of their ribo-seq data, determine the strength of the triplet periodicity signal, generate meta-gene ribosome profiles as well as analyze the relative impact of mRNA sequence features on local read density. RiboGalaxy is accompanied by extensive documentation and tips for helping users. In addition we provide a forum (http://gwips.ucc.ie/Forum) where we encourage users to post their questions and feedback to improve the overall RiboGalaxy service.

Stochastic model of homogeneous coding and latent periodicity in DNA sequences

The concept of latent triplet periodicity in coding DNA sequences which has been earlier extensively discussed is confirmed in the result of analysis of a number of eukaryotic genomes, where latent periodicity of a new type, called profile periodicity, is recognized in the CDSs. Original model of Stochastic Homogeneous Organization of Coding (SHOC-model) in textual string is proposed. This model explains the existence of latent profile periodicity and regularity in DNA sequences.

The use of duplex-specific nuclease in ribosome profiling and a user-friendly software package for Ribo-seq data analysis.

Ribosome profiling is a technique that permits genome-wide, quantitative analysis of translation and has found broad application in recent years. Here we describe a modified profiling protocol and software package designed to benefit more broadly the translation community in terms of simplicity and utility. The protocol, applicable to diverse organisms, including organelles, is based largely on previously published profiling methodologies, but uses duplex-specific nuclease (DSN) as a convenient, species-independent way to reduce rRNA contamination. We show that DSN-based depletion compares favorably with other commonly used rRNA depletion strategies and introduces little bias. The profiling protocol typically produces high levels of triplet periodicity, facilitating the detection of coding sequences, including upstream, downstream, and overlapping open reading frames (ORFs) and an alternative ribosome conformation evident during termination of protein synthesis. In addition, we provide a software package that presents a set of methods for parsing ribosomal profiling data from multiple samples, aligning reads to coding sequences, inferring alternative ORFs, and plotting average and transcript-specific aspects of the data. Methods are also provided for extracting the data in a form suitable for differential analysis of translation and translational efficiency.

Study of triplet periodicity differences inside and between genomes.

Triplet periodicity (TP) is a distinctive feature of the protein coding sequences of both prokaryotic and eukaryotic genomes. In this work, we explored the TP difference inside and between 45 prokaryotic genomes. We constructed two hypotheses of TP distribution on a set of coding sequences and generated artificial datasets that correspond to the hypotheses. We found that TP is more similar inside a genome than between genomes and that TP distribution inside a real genome dataset corresponds to the hypothesis which implies that a common TP pattern exists for the majority of sequences inside a genome. Additionally, we performed gene classification based on TP matrixes. This classification showed that TP allows identification of the genome to which a given gene belongs with more than 85% accuracy.

An Exploration of the Triplet Periodicity in Nucleotide Sequences with a Mature Self-Adaptive Spectral Rotation Approach

Previously, for predicting coding regions in nucleotide sequences, a self-adaptive spectral rotation (SASR) method has been developed, based on a universal statistical feature of the coding regions, named triplet periodicity (TP). It outputs a random walk, that is, TP walk, in the complex plane for the query sequence. Each step in the walk is corresponding to a position in the sequence and generated from a long-term statistic of the TP in the sequence. The coding regions (TP intensive) are then visually discriminated from the noncoding ones (without TP), in the TP walk. In this paper, the behaviors of the walks for random nucleotide sequences are further investigated qualitatively. A slightly leftward trend (a negative noise) in such walks is observed, which is not reported in the previous SASR literatures. An improved SASR, named the mature SASR, is proposed, in order to eliminate the noise and correct the TP walks. Furthermore, a potential sequence pattern opposite to the TP persistent pattern, that is, the TP antipersistent pattern, is explored. The applications of the algorithms on simulated datasets show their capabilities in detecting such a potential sequence pattern.

Поиск парных точек сдвига фазы триплетной периодичности в генах из 17 бактериальных геномов

This paper presents a mathematical method developed for searching for pair phase shifts of triplet periodicity. Such phase shifts could be potential frameshifts in genes resulting from insertions of quite long DNA fragments. We developed software based on the proposed mathematical method and checked if there were pair phase shifts of triplet periodicity in genes of 17 bacteria genomes. The results show that in these 17 genomes about 1 percent of bacteria genes have pair phase shifts of triplet periodicity. This paper also describes a method developed for visualization of pair phase shifts of triplet periodicity and gives examples of such shifts. The research results were partially confirmed by the search for similarities in amino acid sequences that had been made by alternative reading frames. The connection between pair phase shifts of triplet periodicity and frameshift in genes is discussed.

Observation of dually decoded regions of the human genome using ribosome profiling data

The recently developed ribosome profiling technique (Ribo-Seq) allows mapping of the locations of translating ribosomes on mRNAs with subcodon precision. When ribosome protected fragments (RPFs) are aligned to mRNA, a characteristic triplet periodicity pattern is revealed. We utilized the triplet periodicity of RPFs to develop a computational method for detecting transitions between reading frames that occur during programmed ribosomal frameshifting or in dual coding regions where the same nucleotide sequence codes for multiple proteins in different reading frames. Application of this method to ribosome profiling data obtained for human cells allowed us to detect several human genes where the same genomic segment is translated in more than one reading frame (from different transcripts as well as from the same mRNA) and revealed the translation of hitherto unpredicted coding open reading frames.