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Repetitive Dna And Next-generation Sequencing Computational Challenges And Solutions Pdf

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Next-generation sequencing NGS technologies have fostered an unprecedented proliferation of high-throughput sequencing projects and a concomitant development of novel algorithms for the assembly of short reads. However, numerous technical or computational challenges in de novo assembly still remain, although many new ideas and solutions have been suggested to tackle the challenges in both experimental and computational settings. In this review, we first briefly introduce some of the major challenges faced by NGS sequence assembly.

DNA sequencing is the process of determining the nucleic acid sequence — the order of nucleotides in DNA. It includes any method or technology that is used to determine the order of the four bases: adenine , guanine , cytosine , and thymine. The advent of rapid DNA sequencing methods has greatly accelerated biological and medical research and discovery. Knowledge of DNA sequences has become indispensable for basic biological research, and in numerous applied fields such as medical diagnosis , biotechnology , forensic biology , virology and biological systematics. Comparing healthy and mutated DNA sequences can diagnose different diseases including various cancers, [3] characterize antibody repertoire, [4] and can be used to guide patient treatment.

Repetitive DNA and next-generation sequencing: computational challenges and solutions

Metrics details. Repetitive DNA motifs — not coding genetic information and repeated millions to hundreds of times — make up the majority of many genomes.

Fluorescence in situ hybridization FISH -based karyotypes are developed to understand chromosome and repetitive sequence evolution of common oat. The Avena species are monophyletic, but both bioinformatic comparisons of repeats in the different genomes, and in situ hybridization to metaphase chromosomes from the hexaploid species, shows that some repeat families are specific to individual genomes, or the A and D genomes together.

Notably, there are terminal regions of many chromosomes showing different repeat families from the rest of the chromosome, suggesting presence of translocations between the genomes.

The relatively small number of repeat families shows there are evolutionary constraints on their nature and amplification, with mechanisms leading to homogenization, while repeat characterization is useful in providing genome markers and to assist with future assemblies of this large genome c. The frequency of inter-genomic translocations suggests optimum strategies to exploit genetic variation from diploid oats for improvement of the hexaploid may differ from those used widely in bread wheat.

Genome evolution involves multiple processes including whole genome duplications WGDs or polyploidy , segmental genome deletions or duplications, chromosome restructuring fusion, fission, translocation, and inversion , and amplification or loss of gene and repetitive sequences, along with DNA mutation [ 1 , 2 ]. There is a growing interest in reconstructing ancestral genomes of fungi [ 3 ], animals [ 4 ] and plants [ 5 ], revealing principles governing genome evolution and diversification leading to speciation and adaptation.

Repeat motifs vary extensively in sequence and dispersion patterns [ 6 , 7 , 8 ]. Their presence and similarity, variation in copy number and sequences, pose a major challenge to genome assembly and gene annotation [ 9 ].

Repetitive DNA has been postulated to have multiple roles in the genome, including genome stability, recombination, chromatin modulation and modification of gene expression [ 7 ].

Through the decades up to , repeatome knowledge came largely from DNA annealing experiments, screens of random clones, restriction fragment analyses, or amplification of conserved elements with primers. Now whole-genome shotgun sequencing approaches can be used for genome-wide, unbiased repeat analysis [ 11 , 12 , 13 ]. A k-mer analysis counts the number of motifs k-bases long in whole-genome sequence reads [ 14 ], to identify abundant motifs without using reference genomes.

The graph-based clustering analysis e. RepeatExplorer [ 11 , 15 ] is another approach to identify and classify repeats from raw reads. Both are de novo identification strategies, and results can be used for repeat identification or protein domain searches. Because of the multiple genomic locations and difficulties of assembly, in situ hybridization to chromosomal preparation is essential to identify the genomic locations and specificity of repetitive motifs [ 16 ].

These approaches have been used to quantify the genome repetitive landscape in banana, radish, soybean and tobacco [ 17 , 18 , 19 , 20 , 21 ]. Common oat Avena sativa L. Genomic resource development of common oat, important for breeding and improvement, has lagged behind other major crops [ 24 , 25 ].

The oat genome contains numerous families of repeats and apparently frequent chromosome translocations [ 29 , 30 ]. In recent phylogenetic analyses, common oat was inferred to experience ancient allotetraploidy and recent allohexaploidy events involving C-, A- and D-genome ancestors [ 31 , 32 ], while the genome reshuffling obscures contributions of different candidate maternal A-genome progenitors bipaternal genome definition referred to [ 32 ].

Here, we aimed to elucidate structure, organization, and relationship of all major repetitive DNA classes in diploid and hexaploid oats, examine their chromosomal locations, and understand the significance of repeatome in genome and chromosome evolution of Avena in the context of genomic, bioinformatic and cytogenetic evidence.

The complete picture of repetitive DNAs provides new evidence for events occurring during evolution and speciation in the genus, including hybridization and chromosomal translocation events. For graph-based clustering of reads, a 1. Only a small proportion 2. Our analyses were not designed to identify most microsatellite arrays including telomeric sequences , typically shorter than mers. Clusters showed characteristic graph patterns Fig. As examples, 0.

For cumulative repetitivity frequency plots of to mers, the steeper slope indicated the faster cumulative percentage changes, which varied relatively gentle for short k-mers to mers and gradually increased steep-slope for longer k-mers to mers Fig. For the same repetitivity frequency, a shorter k-mer motif has a higher cumulative percentage and higher frequency in raw reads; Fig.

Overall, the graphs were consistent with the RepeatExplorer analysis, with a group of very abundant sequences representing about a quarter of the genome inflection in e. To localise repeats on Avena sativa chromosomes Figs. Copy numbers and relative proportion of the selected probes were analysed in silico in A.

The monomer number shown in dotplots Fig. Nanopore or PacBio Sequel or chromosome walking e. BAC clones. Repeat copy numbers in the three diploid A genome species analysed was not the same Fig. No repeat was predominant in A. One repeat family Ast-R was much more abundant in A. The Asmer43bp repeat was only abundant in A. Results from in situ hybridization of 25 repetitive sequences identified here to A.

Chromosomes were numbered by descending order of sizes and arranged in pairs using morphology and hybridization patterns: chromosomes 1—14, 15—28 and 29—42 belong to C-, A-, D-genomes, respectively Figs. Both in situ hybridization patterns and bioinformatic copy number counting allowed us to classify repeat sequences into five categories depending on genome specificity: C-, A-, and D-genome specific repeats showed stronger hybridization to chromosomes of one genome Fig.

Ten non-homologous in situ hybridization signals were detected at intercalary, pericentromeric, and subtelomeric regions on 14 C-genome-origin chromosomes. As-T and As-T Fig. Retrotransposon Ast-R Fig. Ast-R showed D-C translocations with subterminal double-dot signals on 12 D-chromosomes 29—40; Additional file 4 : Figure S4a-S4f , but missing terminal signals on 12 D-chromosomes 29—40 that in turn show C genomic repeats Fig.

Similarly, Ast-T was missing from 12 D-chromosome long arm terminals chromosomes 29—40; Additional file 5 : Figure S5a-S5f that either showed A- or C-genome-specificity. Ast-T showed typical signals of tandem repeats, i. Tandem repeat Ab-T Fig. Combining the in silico analysis with molecular cytogenetics on chromosomes in situ, we could identify the nature of the motifs and measure their abundance to give a comprehensive survey and evolutionary relationships of the repeat landscape of oat Figs.

Our strategy would not expect to reveal microsatellite motifs, short runs of dinucleotide or trinucleotide repeats with unique flanking regions, known to have an uneven distribution across the genome [ 44 ]. While there are increasing reports of genome-wide repeat surveys [ 13 , 45 , 46 ], most sequence assemblies collapse repeats to variable extents [ 21 , 47 ], while library screening or PCR amplification with primers are selective.

Thus detailed comparisons between our results and many published analyses using whole genome assemblies, reference repeats e. RepeatMasker , or targeted screening may not be valid. Many of the major families of repeats identified here have been identified previously in selective screens of DNA libraries [ 30 , 36 ], although these studies could not quantify their abundance in the various diploid and the hexaploid genomes.

Importantly, unlike the analysis of unprocessed random reads here, selective screens cannot show that all the repetitive components of the genome have been surveyed. Frequency of major repetitive DNA classes in Avena sativa. Repeats were identified by graph-based clustering RepeatExplorer and in abundant k-mer motifs, and classified by nucleotide domain hits and database Blast searches.

Repeat cluster graphs and dotplots of selected repeats in Avena species. For each repeat, the RepeatExplorer cluster is shown left; yellow nodes represent all assembled contigs within the repeat cluster; red nodes represent members of the contig analysed in greater detail, including by amplification and in situ hybridization.

A self-dotplot of the selected contig is shown in the right panel; parallel diagonals show tandem repeats. Repeat names include species origin of the exemplar family member: Ab, Avena brevis ; Ah, A.

The mers identified by our k-mer analysis with more than 10 copies per genome correspond to the figures from potato and tomato see Fig. A change in slope, as seen in A.

LTR retrotransposons are largely responsible for the dramatic differences in genome sizes between related plant species, e. K-mer repetitivity frequencies in Avena genome raw reads. Cumulative percentages of k-mer motifs are plotted against frequencies of different k-mers in Avena sativa a , A.

Comparison of mer d and mer e frequencies in four Avena genomes. Cumulative mer frequency f of Avena genomes in comparison with Petunia axillaris Bombarely et al. These values are closely similar to the three wheat genomes ratio 2. DNA transposons, shorter in element length than retroelements, represented 5. All genomes have mechanisms controlling TE amplification.

Schorn et al. Large genomes bear higher proportions of TE sequence, and Lyu et al. Here, it is notable that oat retrotransposon-related repetitive sequences families vary in abundance between diploid species, and some are essentially specific to one or two of the genomes Fig.

Tandem repeats or satellite DNA is a feature of most eukaryotic genomes. Structural interactions between nucleosomes and DNA repeats can impact chromatin dynamics [ 58 , 59 ] and the stable wrapping of tandem repeats could be important for genome stability and methylation of domains leading to silencing. Submotifs of a repeat family can be used as genome-specific probes for in situ hybridization, e. They are a relatively unusual length for plant repeat motifs, although c.

Localization of repetitive sequences on A. White asterisks, arrows, and arrowheads indicate notable C-, A-, and D-chromosome signals. Details of colours and arrangements as Fig. Relative proportions and an evolutionary model of repetitive DNA motifs in common oat genomes. Ab, A. T, tandem; R, retrotransposon. Based on phylogenetic evidence, in Avena , two NORs 45S rDNA sites per haploid chromosome set were ancestral characters, while chromosome complements with 4 or more NORs were derived characters [ 63 ].

The karyotypes with repetitive sequence locations provide a fresh perspective in understanding evolution in Avena. A-genome specific pAsa was isolated long ago [ 36 ]. They discussed the repeat length and existence of four monomers inserted within pAsa, suggesting cautiously that the pAsa sequence could be classified as a satellite DNA sequence.

However, twenty years later, we still share the uncertainty of Linares et al. Other sequence families also show differential amplification or reduction in individual Avena A-genomes Fig.

As-T or high abundance of D-chromosome specific motifs identified in A. This is supported by greater proportion of C-genome specific motifs, diverging from the common ancestors before the radiation of A- and D-genome specific motifs, as the A- and D-genome specific motifs amplified independently in common oat Fig.

This evolutionary scenario is also supported by repeats common to the A- and D-genomes or all three genomes, but no repeats were found to be specific for the C- and A- or C- and D- genomes. Retrotransposons may have a role in genome behaviour by acting as nuclei for RNA-dependent DNA methylation as [ 65 ] , leading to position effect variegation via heterochromatinization around repetitive elements affecting adjacent gene expression [ 66 , 67 ].

Next Generation Sequencing in Aquatic Models

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Erratum: Repetitive DNA and next-generation sequencing: computational challenges and solutions

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Пока файл Цифровой крепости не подменен модифицированной версией, копия ключа, находившаяся у Танкадо, продолжает представлять собой огромную опасность. - Когда мы внесем эту поправку, - добавил Стратмор, - мне будет все равно, сколько ключей гуляет по свету: чем их больше, тем забавнее.  - Он жестом попросил ее возобновить поиск.

Технология развивается в геометрической профессии, и рано или поздно алгоритмы, которыми пользуется общество, перестанут быть надежными. Понадобятся лучшие алгоритмы, чтобы противостоять компьютерам завтрашнего дня. - Такова Цифровая крепость. - Конечно. Алгоритм, не подающийся грубой силе, никогда не устареет, какими бы мощными ни стали компьютеры, взламывающие шифры.


Georgette D. 31.05.2021 at 15:51

Background: Next-generation sequencing NGS technologies have fostered an unprecedented proliferation of high-throughput sequencing projects and a concomitant development of novel algorithms for the assembly of short reads.

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The most valuable application of next generation sequencing NGS technology is genome sequencing.

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Repetitive DNA and next-generation sequencing: Computational challenges and solutions. November ; Nature Reviews Genetics.

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