Disadvantage of Short Reads in Genome Assemby
Genome Assembly
For example, if a genome associates is finished to the level of whole linear chromosomes, the ends will contain tandem (consecutive) repeat sequences institute inside telomeres, ranging from 5-mer to 27-mer repeated several thou times, which both protect the end of the chromosome from deterioration, chromosomal fusion, or recombination, and every bit a machinery for senescence and triggering apoptosis.
From: Advances in Genetics , 2017
A Primer of Molecular Biology
Betsy Foxman , in Molecular Tools and Communicable diseases Epidemiology, 2012
5.10.2 Factor Associates
Genome assembly refers to the process of putting nucleotide sequence into the correct order. Associates is required, because sequence read lengths – at least for at present – are much shorter than most genomes or even near genes. Genome assembly is made easier by the existence of public databases, freely available on the National Center for Biotechnology Information website ( http://www.ncbi.nlm.nih.gov). Just every bit it is much easier to assemble a moving picture puzzle if you know what the picture looks like, information technology is much easier to get together genes and genomes if you have a skilful idea of the sequence order. In the human genome, genes occur in the same physical location on the chromosome, simply there tin be dissimilar numbers of copies and variable numbers of repeated sequence that complicate assembly. Although bacterial genomes are much smaller, genes are not necessarily in the aforementioned location and multiple copies of the aforementioned gene may announced in different locations on the genome. Therefore even with the availability of commercial software and ever growing reference databases, the process of genome associates tin can accept considerably longer than the time to obtain actual sequence.
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The new era of genome sequencing using high-throughput sequencing technology: generation of the get-go version of the Atlantic cod genome
Ole Kristian Tørresen , ... Alexander J. Nederbragt , in Genomics in Aquaculture, 2016
Goals for a new reference genome
Genome assemblies represent models for the actual genome—and thus are never perfect. A unmarried associates cannot represent all the diversity within populations of a species, and information technology is well-nigh impossible to eliminate all possible technological or algorithmical errors. Therefore, published genomes that have an agile research community are continuously improved. For case, in Dec. 2013, a new version of the human genome associates was released (build 38), with several improvements compared to build 37, showtime released in 2009.
The beginning version of the cod genome has proven to be a valuable resources for the fish genomics community, and is frequently cited and downloaded. The fragmented nature of the Atlantic cod genome associates withal, poses limitations compared to some of the other available teleost genomes. For case, synteny analysis was only possible to a limited extent, since not all scaffolds have been placed within the context of a larger chromosomal region. Moreover, the current number of gaps in the scaffolds results in the presence of relatively big parts of the genome that are solely bachelor as short, unplaced pieces. Nosotros therefore continuously work toward an improved version of the genome (encounter Chapter 3).
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Additional Bioinformatic Analyses Involving Nucleic-Acid Sequences*
Supratim Choudhuri , in Bioinformatics for Beginners, 2014
seven.two Sequence Associates
Genome assembly from sequence reads is an algorithm-driven automated procedure. DNA-sequence-associates programs take utilized sequence overlaps for sequence assembly in correct order. The computational aspect of assembly algorithms is beyond the scope of this book. Still, a few terms will be discussed in patently language for the sake of familiarity. Sequence assembly can be washed using one of three approaches: (1) greedy, (2) overlap-layout-consensus (OLC) and Hamiltonian path, and (3) de Bruijn graph and Eulerian path d .
Greedy is a rapid-associates algorithm, which joins together the sequence reads that are the nearly like to each other based on equally much sequence overlap as possible. In doing so, the greedy algorithm offset compares all fragments in a pairwise style to identify sequences that take overlaps; adjacent, the sequences that have the all-time overlaps are merged; this merging process continues (iterative process) until all the sequences with overlaps accept been merged. In this procedure, some reads may not be assembled, which are shown as gaps. Paired-cease sequencing is used to close the gaps. Many early assemblers were based on the greedy algorithm and were extremely useful, such equally Phrap, TIGR assembler, and CAP. The Phred–Phrap–Consed suite of programs has been widely used. Phred and Phrap were developed by Drs Phil Green and Brent Ewing at the University of Washington, Seattle, in 1998 for the Human being Genome Sequencing project. Phred is base-calling software that assigns a quality score to each base called. Phrap is de novo shotgun sequence-assembly software. Consed is the sequence-assembly editor companion to Phrap, and it is a tool for viewing, editing, and finishing sequence assemblies created with Phrap. Many such assembly suites also include sequence-alignment tools.
The overlap-layout-consensus (OLC) algorithm is based on all pairwise comparisons, and it generates a directed graph using reads and overlaps east . In the graph, each sequence is created as a node and an edge is created betwixt any two nodes whose sequences overlap. The algorithm then tries to find the Hamiltonian traversal path of the graph, which contains all the nodes (sequences) exactly once, and combines the overlapping sequences in the nodes into the sequence of the genome. Some assemblers that utilize the OLC algorithm are Arachne, CABOG (Celera Assembler), Newbler, Minimus, Edena, and MIRA. Overlap-based approaches have been generally used for longer reads (>200 bp). Notwithstanding, overlap-based assemblers for brusque reads have also been developed. 4
The de Bruijn–graph-based approach has been successfully employed in assembling short reads (<100 bp). However, de Bruijn graph assemblers have also been successfully used with longer reads. iv Some assemblers that utilize the de Bruijn–graph algorithm are Euler-SR, Oases, Velvet, ALLPATH, ABySS, and SOAPdenovo. Sequence assembly based on meaning sequence overlap, as washed using the standard Sanger method, works well when at that place are a finite number of sequence reads to exist assembled. However, adjacent-gen sequencing generates hundreds of millions of sequence reads. The assembly of such a big number of sequence reads cannot be done easily using this traditional method. The problem of scalability is solved by using the de Bruijn graph. The de Bruijn graph does not use the actual sequence reads for assembly, but breaks each sequence read downwardly to smaller sequences called m-mers. These yard-mers are aligned using (k−1) sequence overlaps. The actual size of g depends on sequence coverage, read length, etc., just normally is non less than half of the bodily read length. For example, a 106-base read tin be divided into 49 overlapping 58-mers (sequence read length−one thousand-mer length+ane=# of grand-mers; hence, 106−58+one=49). Because breaking ane sequence read into k-mers increases the number of curt sequence reads (e.g. just 1 106-base read generates 49 k-mers, each i 58 bases long), information technology is likely that the resulting k-mers generated from all sequence reads will represent about all k-mers from the genome for sufficiently small k. This procedure seemingly compensates for missing sequence reads—that is, the sequence reads that could not be generated through sequencing for a multifariousness of technical reasons. 5 Therefore, computational awarding of the de Bruijn graph helps alleviate many problems of de novo sequence assembly, simply information technology is still not a fool-proof process.
With the comeback of sequence coverage and computing ability, software is being constantly beingness developed or improved based on newer algorithms. Sequence reads can now be accurately assembled based on overlaps as small as 15 bp. 6
A genome sequence assembly can be performed in two ways: mapping and assembly, or de novo assembly. If the genome has been sequenced before and a reference genome sequence already exists, and then the newly obtained resequence reads are showtime mapped to the reference genome through alignment and then assembled in proper lodge; this mode of associates is called "mapping and assembly." Bowtie is an ultrafast, memory-efficient brusque-read aligner that helps in mapping and associates. It apace aligns large sets of short sequencing reads to a reference sequence, at a rate of over 25 meg 35-bp reads per hr. For reads longer than nigh 50 bp, Bowtie 2 is by and large faster, more sensitive, and uses less retentiveness than the original Bowtie (http://bowtie-bio.sourceforge.net/index.shtml).
In contrast, if there is no reference genome sequence then the assembly is chosen "de novo associates." For de novo assembly, paired reads work amend than single reads considering paired reads assistance generate scaffolds. Therefore, genome associates is a hierarchical procedure; it is performed in steps beginning from the assembly of the sequence reads into contigs, assembly of the contigs into scaffolds (supercontigs), and associates of the scaffolds into chromosomes. Many genome assemblies remain restricted to scaffold level for a long time because the gaps can non be easily sequenced. Some scaffolds can be placed inside a chromosome, while the chromosomal assignment of other scaffolds may remain difficult.
The de novo genome assembly can be assessed based on a number of parameters, such every bit the number of contigs and scaffolds available and their size, and the fraction of reads that can be assembled. One widely used metric to evaluate the quality of associates is the contig and scaffold N50 value (see Box 7.ane). An N50 contig is the size of the shortest contig such that the sum of contigs of that size or longer constitutes at least 50% of the full size of the assembled contigs. For case, an N50 contig of 100 kb ways that when contigs of 100 kb or longer are added upwards, the resulting size represents at least 50% of the total size of all assembled contigs. Also, an N50 scaffold size is the length of the shortest scaffold such that the sum of the scaffolds of that size or longer constitutes at least 50% of the total size of all assembled scaffolds.
Box 7.ane
The N50 contig value can be determined by first sorting all contigs in decreasing order of size, and so adding the contigs until the total added size reaches at to the lowest degree half of the full size of all assembled contigs. The size of the smallest contig used in this improver procedure represents the N50. The scaffold N50 is calculated in the aforementioned mode using the scaffold size. For example, if the contigs assembled are 0.43, 0.75, 1, 0.6, 0.viii, 0.55, 0.32, and 0.25 Mbp, the total assembled size of all contigs is four.vii Mbp. Now, organizing the contigs in decreasing gild of size, we go: 1, 0.8, 0.75, 0.6, 0.55, 0.43, 0.32, and 0.25 Mbp. Adding just 1, 0.8, and 0.75 yields 2.55 Mbp, which is 54% of the total assembled size of all contigs. The smallest contig used in this add-on procedure is 0.75 Mbp. Therefore, the N50 contig is 0.75 Mbp. The larger the N50 value, the ameliorate is the assembly. Using the aforementioned concept, college values of N are also used, such as N60 and N80. If the N50 scaffold length is too short, additional rounds of shotgun sequencing are recommended.
Although genome sequencing has go high throughput and very cheap, and the computational power in genome-sequence assembly has tremendously increased, the electric current methods have many problems, partly owing to the nature of the genome sequence itself and partly owing to bug inherent in the sequencing method. Consequently, de novo sequence assembly is all the same a major challenge and tin can be fraught with errors and missing sequence. 7 This makes finishing a genome sequence and assembly a continuous and long-drawn-out process.
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Silkworm genomics
Manjunatha H. Boregowda , in Advances in Animal Genomics, 2021
16.8.iv.2 Bombyx genome new associates and its characteristics
The new genome assembly size as estimated subsequently resequencing the genomic DNA using PacBio long-read and Illumina short-read sequencer, is 460.3 Mb, which varies with the previous WGS data and sequence data is available at SilkBase (http://silkbase.ab.a.u-tokyo.air-conditioning.jp, Kawamoto et al., 2019). This hybrid assembly has 37.3 Gb PacBio reads (equal to about 80 × coverage), and 27.9 Gb pairs of Illumina reads (virtually lx ×), and the resultant combined total coverage is about 140 ×). The new genome assembly posses 696 scaffolds with an N50 size of 16.eight Mb, while the longest scaffold and contig N50 are 21.5 and 12.2 Mb, respectively. With the genome information available for 24 Lepidopteran insects in Lepbase (http://lepbase.org), information technology is observed that the B. mori new genome assembly is one of the best reference sequences.
Comparatively, RNA-seq reads from dissimilar tissues of B. mori, brain, early embryo, epidermis, fat body, internal genitalia, midgut, anterior silk gland, and eye silk gland, which are mapped onto the new and old genome assemblies, signal that the new genome assembly covers wider transcriptionally active regions than the one-time genome associates of the B. mori genome. In addition, TEs, which are the source of piRNAs, known to interfere in the cosmos of accurate genome assembly and more often than not constitute in the gap regions between the old genome scaffolds, are mapped on to both new and quondam assemblies. These piRNAs reads, from xi bachelor libraries (0, 6, 12, 24, and 40 h post-fertilization eggs, ovaries from three strains, testes from two strains, and an ovarian prison cell line BmN4), are mapped to wider range on the new genome assembly than the former assembly of the B. mori genome. This shows that repeat regions are precisely assembled using the long-read sequencer that resulted in a remarkable decrease in the number of piRNA clusters and an intern increase the total length, average length, median length, and N50 of the new assembly compared to previous genome assemblies. In addition, a number of transposable elements are also newly identified. With this, currently, the Bombyx new genome assembly appears to exist a much meliorate genome with higher coverage compared to previous genome sequence assemblies.
Although, to achieve high-quality genome sequence assembly and the gene count, deep sequencing of short (Illumina) and long (PacBio) reads is performed using the silkworm strain p50T (Daizo), merely such equivalent assembly information is however lacking from Chinese silkworm strain p50 (Dazao) for comparative analysis and to obtain all the same more high-quality genome sequence assembly for B. mori.
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Fungal Genomics
Brendan Loftus , in Applied Mycology and Biotechnology, 2003
three.4 Genome Assembly of Fungal Genomes
Whole genome associates draws together unique portions of the genome equally an initial step, and characterize sequentially, the remaining hard-to-get together regions based on the bachelor testify. This reduces the overall errors in the private assemblies to a minimum, while producing the most authentic draft of the overall structure of a genome. Accurate computational assembly of the fungal genomes currently underway should not testify a major technical hurdle, given the demonstrated ability of the assemblers to assemble the human, and other large eukaryotic genomes. Available assembly data from a various array of fungal genomes including Cryptococcus, Neurospora, Magnaporthe, Aspergillus and Coccidoides, indicates that this is indeed the case. In this context, the genome of Candida albicans may be considered an exception as its genome is diploid and standard sequence associates software does non recognize the possibility of diploidy. Therefore, when confronted with sufficiently unlike alleles, the assembler often assembles them into separate contigs. This problem may become more significant as more diploid or polyploid fungi are sequenced in the hereafter. A number of the above projects accept supplemental mapping information which proves to be a groovy cross referencing method for the veracity of computational assemblies. In general, the presence of a physical map is a useful resource but as sequencing applied science is progressing and so chop-chop relative to map availability, the latter is unlikely to exist a resource for many future fungal genome projects. Indeed, given the demonstrated benefits of the increased use of clone constraint information from insert libraries of unlike sizes in computational associates, the presence of a physical map may not be deemed an essential component for the correct assembly of fungal genomes going forrard.
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DNA Sequencing for the Detection of Human being Genome Variation
Samuel Levy , Yu-Hui Rogers , in Essentials of Genomic and Personalized Medicine, 2010
Whole Genome Associates
The whole genome assembly process utilizes several aspects of experimental design to ensure that unambiguous construction of long contiguous sequence can be generated. These include the generation of sequence reads from each end of clone inserts using universal primers pairs to ensure that a majority of inserts volition have their ends sequenced. This enables both the generation of contiguous sequence and the potential for ordering and orienting larger sequence segments substantially created by sequence alignment of the individual reads whose mate pair relationship is known. A multifariousness of genome assembler software packages have been designed with this basic rationale at their core (Celera Associates (Myers et al., 2000), Phusion (Mullikin and Ning, 2003)). The Atlas assembler uses equally input both BAC-based clone sequences and reads generated via the WGS strategy (Havlak et al., 2004), an arroyo that was used to assemble the rat genome (Gibbs et al., 2004).
There are two basic steps to assembly: (i) the creation of unique regions of contiguous (contigs) assemblies from the sequence overlaps between reads and (ii) the sequential stop-to-cease organization of contigs past employing the mate pair information contained in the reads constituting each contig. The successful application of this strategy was a significant challenge for the sequencing and assembly of human Deoxyribonucleic acid since greater than 45% of the genome sequence is repetitive in nature (Lander et al., 2001; Venter et al., 2001). The repetitive nature of the human and other mammalian genomes thus confounds the ability to determine accurate read sequence overlaps and the placement of contigs into the correct order and orientation. Our solution is to employ clone insert libraries of a size larger than the corresponding repeat regions, typically either fosmid or BAC libraries (40 kb and >100 kb respectively), thus enabling the spanning of repeats past assembling next unique sequence (Venter et al., 2001). The selection of a range of insert sizes libraries is typically congenital into the experimental pattern when sequencing a new genome.
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Avian genomics
Hans H. Cheng , ... Huaijun Zhou , in Sturkie's Avian Physiology (Seventh Edition), 2022
ii.3.iii Genes
The original chicken genome assembly estimated there were twenty,000–23,000 protein-coding genes ( International Chicken Genome Sequencing Consortium, 2004). However, more recent estimates from the comparison of 48 avian genomes indicates the number for bird genomes is lower at 15,000–16,000 (Zhang et al., 2014). Based on this number, this would reflect a ∼30% reduction compared to the number establish in mammals. At least 1241 of the loss in genes can exist explained by large segmental deletions during the evolution of birds. However, ∼lxx% of the lost genes bear witness paralogs suggesting functional compensation. In improver, some of the gene loss may not exist truthful but rather the inability to detect genes in high GC-rich regions (Bornelov et al., 2017).
Avian genes are ∼50% smaller than their mammalian counterparts, mainly due to the shortening of introns and reduced distances between genes, which helps to account for the reduced size of avian genomes. Interestingly, with respect to avian-specific highly conserved elements, most are significantly associated with transcription factors associated with metabolism. Having a complete list of genes in many avian species has also enabled hypotheses on the evolution of flight, diets, vision, and reproductive traits (Zhang et al., 2014).
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PeanutBase and Other Bioinformatic Resources for Peanut
Sudhansu Nuance , ... Steven B. Cannon , in Peanuts, 2016
Accessing and Using the Genome Assemblies and Genes
The ii diploid genome assemblies (A. duranensis and A. ipaënsis) tin can be explored at PeanutBase through genome browsers implemented in GBrowse (Stein, 2013). The predicted gene models for both species are included as tracks, along with cistron models for soybean and mutual edible bean and syntenic regions in soybean, common edible bean, and Medicago truncatula (Figure 3). Each of these features link to the corresponding features on other browsers, due east.g., the Phaseolus genes on the Arachis browsers link to genes at the Phaseolus browser at http://legumeinfo.org, and the soybean synteny tracks lead to the respective regions at the Glycine max (L.) Merrill browser at http://soybase.org (Figure iv).
For sequence searching, a BLAT (Kent, 2002) utility is available through GBrowse and through a carve up BLAT sequence–search interface. Sequence searching is also available through a BLAST (Camacho et al., 2009) interface developed for PeanutBase, the Tripal Boom module. This module was developed in collaboration with the Tripal development customs and is in apply by a number of different Tripal-based websites.
FASTA downloads of the assembly, raw and repeat masked, along with downloads of the individual scaffolds that were assembled to make the chromosomes are available at PeanutBase. The scaffolds are likewise available from GenBank's Whole Genome Shotgun database (accessions JQIN00000000 for A. duranensis and JQIO00000000 for A. ipaënsis).
Gene models are available for download, searching, and browsing at PeanutBase. At that place are two sets of gene models for each species: a main analysis set recommended by the PGC for near analyses and another set that is available for exploring culling gene structures.
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Inference of Horizontal Gene Transfer: Gaining Insights Into Evolution via Lateral Conquering of Genetic Material
Suhaila Sulaiman , ... Mohd Firdaus-Raih , in Encyclopedia of Bioinformatics and Computational Biological science, 2019
Identification of HGT Events Using Transcriptome Data
Since de novo whole genome assembly of a plant genome is complicated by the presence of repeat sequences, assessing HGT at the transcriptome level provides a direct approach to reveal the expression status of the genes. In host-parasite systems, a gene is considered horizontally caused if it is phylogenetically placed nearer to its host rather than to its closest relatives. HGT detection using software is fabricated possible for plants with the development of various tools, such equally T-rex ( Boc et al., 2012), Alienness (Rancurel et al., 2017) and HGTector (Zhu et al., 2014). Nevertheless, some of these tools require specific files and preparation steps to conduct the HGT search. For instance, 2 newick trees generated for species and gene of interest are required as the input files. Every bit with Alienness, a BLAST file of a whole proteome of interest is required to blast confronting any NCBI poly peptide library. This tool calculates an Alien Index (AI) for early query protein and an AI>0 indicates a possible HGT. HGTector takes one or more protein sets as input. In principle, this tool formulates a grouping scenario, calculates the 3 weights of each cistron, defines certain cutoffs and and so identifies the genes that exhibit singular distribution.
In a case written report that involves a parasitic found (Section HGT in Plants), 2 web servers, OrthoVenn (meet "Relevant Websites section") (Wang et al., 2015) and eggNOG-mapper of the EggNOG five 4.5 (see "Relevant Websites section") (Huerta-Cepas et al., 2016), were used to cluster orthologous genes beyond Rafflesia (parasite) and Tetrastigma (host) predicted proteomes. Homologous sequences were retrieved for each candidate HGT genes and were aligned using the multiple sequence alignment tool MUSCLE (Edgar, 2004) embedded in MEGA7 (Kumar et al., 2016), which was later on used to perform phylogenetic assay through maximum likelihood searching.
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Insect Transposable Elements
Zhijian Tu , in Insect Molecular Biology and Biochemistry, 2012
3.4.4 Insights from Comparative Genomic Analysis
There are 31 insect genome assemblies available at the National Center for Biotechnology Information (NCBI) Genome Project Database (ncbi.nlm.nih.gov/entrez): nineteen genome assemblies are bachelor from Dipteran species, including i Hessian wing (http://world wide web.ncbi.nlm.nih.gov/genomeprj/45867) and 6 mosquitoes (Holt et al., 2002; Nene et al., 2007; Arensburger et al., 2010; Lawniczak et al., 2010; http://www.ncbi.nlm.nih.gov/genomeprj/46227); and 12 Drosophila (Drosophila 12 Genomes Consortium, 2007). At that place are assemblies from seven Hymenopteran species, including the honeybee (Honeybee Genome Sequencing Consortium, 2006), 3 ants (Bonasio et al., 2010; http://world wide web.ncbi.nlm.nih.gov/genomeprj/48091), and 3 wasps (Werren et al., 2010). There are also assemblies from one Lepidopteran species, Bombyx mori (Xia et al., 2004; Mita et al., 2004), and one Coleopteran species, Tribolium castaneum (Tribolium Genome Sequencing Consortium, 2008). Assemblies from 3 hemimetabolous insects are available, including one Phthiraptern insect body louse (Kirkness et al., 2010) and two Hemipteran (The International Aphid Genomics Consortium, 2010; http://www.ncbi.nlm.nih.gov/genomeprj/13648). In improver, a number of insect genomes are beingness sequenced using "adjacent-generation" approaches, and information technology is predictable that rapid expansion of sequenced genomes volition bring tremendous opportunities to the investigation of TE diversity and evolution. Whole-genome comparative analysis of insect TEs is notwithstanding in its early stages, and a few interesting observations are highlighted below. Systematic analysis of the 12 Drosophila genomes revealed that while the TE content varies from 2.vii% to ~25% of the host genomes, the relative abundance of different groups of TEs is conserved across nigh of the species (Drosophila 12 Genomes Consortium, 2007). Comprehensive analysis identified over 100 potential horizontal transfer events by more twenty TEs among the 12 Drosophila species, nearly of which involved DNA transposons and LTR retrotransposons (Loreto et al., 2008; Bartolome et al., 2009). Systematic comparison of multiple aligned genomes revealed TE insertion sites across the entire genomes, and supported a hypothesis that most TEs in D. melanogaster are recently active (Caspi and Pachter, 2006). The published genomes of Anopheles, Culex, and Aedes mosquitoes vary past 5-fold in size, ranging from ~270 Mbp for An. gambiae (Holt et al., 2002) to ~500 Mbp for C. quinquefasciatus (Arensburger et al., 2010), and ~1300 Mbp for Ae. aegypti (Nene et al., 2007). TE contents in these three species are eleven–16%, 29%, and 47% of the assembled genomes, respectively, indicating that TEs contributed significantly to the genome size variations amongst mosquito species. While sixteen% of the Ae. aegypti genome is occupied by MITE-similar elements, cut-and-paste Deoxyribonucleic acid transposons represent simply iii% of the genome, suggesting that a modest number of DNA transposons may be responsible for cross-mobilizing a large number of non-autonomous MITE-like sequences (Nene et al., 2007). Systematic comparisons also revealed an credible horizontal transfer consequence betwixt Aedes and Anopheles mosquitoes involving an ITmD37E DNA transposon (Biedler and Tu, 2007). Among the sequenced Hymenopteran species, the honeybee genome contains just ~7% repetitive sequences while echo contents range from 15 to 27% in the ants and wasps (Honeybee Genome Sequencing Consortium, 2006; Bonasio et al., 2010; Werren et al., 2010). The parasitic trunk louse harbors simply a very pocket-sized number of TEs, which occupy 1% of its 110-Mbp genome (Kirkness et al., 2010).
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