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Next Generation Complex Genome Assembly

Next Generation Complex Genome Assembly

6 th International Symposium Breeding Research on Medicinal and Aromatic Plants, BREEDMAP 6, Quedlinburg, Germany, June 19-23, 2016 18 Julius-Kühn-Archiv, 453, 2016 ASL 4: Next Generation Complex Genome Assembly Kobi Baruch 1 , Omer Barad 1 , Gil Ben Zvi 1 , Gil Ronen 1

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A candle in the dark: Reference genome assembly for rye highlights the importance of data visualisation and manual editing

A candle in the dark: Reference genome assembly for rye highlights the importance of data visualisation and manual editing

genome for rye, which was completed following the philosophy that the results of automated procedures are best taken as suggestions, to be carefully refined by a human curator with access to an array of intuitive visualisations. Such close curation can markedly increase the quality of a genome assembly, and visually-intuitive representations of a genome assembly (and its relationship to the underlying data), are similarly valuable for those using the genome for downstream applications.

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De novo Nd-1 genome assembly reveals genomic diversity of Arabidopsis thaliana and facilitates genome-wide non-canonical splice site analysis across plant species

De novo Nd-1 genome assembly reveals genomic diversity of Arabidopsis thaliana and facilitates genome-wide non-canonical splice site analysis across plant species

This work described two versions of an A. thaliana Nd-1 de novo genome assembly [53,213]. Although the assembly contiguity was substantially improved by long single molecule real-time (SMRT) sequencing reads, there are still genome regions missing in the second assembly. Almost 20 years after the release of the first A. thaliana genome sequence the currently available genome sequence is still incomplete. Centromeres and nucleolus organising regions (NORs) pose a challenge and require the routine generation of even longer reads or alternatively reads with substantially lower error rates [54,213]. There are first reports of single molecules sequenced via Oxford Nanopore Technologies (ONT) substantially exceeding the 2 Mbp mark [123]. If the read length could be further increased, this technology might have the potential to finally enable the closure of the last remaining gaps in the A. thaliana genome sequence. Improvements of nanopore sequencing e.g. re-reading of the very same DNA strand [75] or coupling of two nanopores with different error profiles [214] might lead to the required improvements of ONT read quality. However, latest improvements of sequencing technologies require improved DNA extraction protocols to provide high molecular input material [213,473,474]. Therefore, the bottleneck in generating even longer reads is likely to be the DNA extraction process. Efficient separation of high molecular DNA molecules from smaller fragments would be required to harness the full potential of long read sequencing technologies.
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Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties

Genome assembly of wild tea tree DASZ reveals pedigree and selection history of tea varieties

Here, we present the genome of an ancient tea tree. Moreover, RNA sequencing of an additional 217 tea accessions is performed to characterize the genetic diversity, population structure, and pedigree relationship among a representative set of Chinese tea germplasms. We also demonstrate the utilization of the genome sequence and diverse natural populations of tea in the identifi- cation of genes and functional variations that regulate the content of catechins and gallic acid (GA) in tea leaves. These resources would facilitate the genetic improvement of tea plants as well as advance our understanding of the biosynthesis of health- beneficial natural products in tea.
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Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases

Tandem repeats lead to sequence assembly errors and impose multi-level challenges for genome and protein databases

well as duplicated (or otherwise multiplied) domains within a protein-coding gene. They are affected by the filtering and masking operations during genome assembly. A prob- lem occurs when the read length of the sequencing method is shorter than the LTR––in this case, repeat numbers can be massively misjudged. In the case of protein-coding re- gions, this has direct effects on the interpretation of biolog- ical function. LTRs are not uncommon in structural pro- teins on cell surfaces, and in pathogenicity factors of bac- teria, parasites, and viruses. As an example, Wrobel et al. ( 81 ) have shown that in the fish pathogen Yersinia ruckeri, a surface adhesin involved in biofilm formation called Ilm has >20 Ig-like domains repeated in tandem that are iden- tical even on the DNA level (repeat length ∼300 bp). Re- peat numbers vary slightly from strain to strain, but in this case only PacBio-based genomes show the correct number of repeats (Figure 1 ). Deposited genomes based on short- read methods show underestimated repeat numbers (by a factor of 4 to 5). The fact that the underestimated repeat number is an approximation made during genome assembly is not visible in the deposited genome data. In a very simi- lar example, Franz´en et al. find that in the human and an- imal parasite Giardia, variable surface proteins (VSPs) are difficult to sequence using 454 sequencing. Using this tech- nology, only a few genes could be assembled due to their highly repetitive nature ( 82 ). From other experiments (in- cluding some re-sequencing using different technologies), the authors estimate that ca. 300 of these repetitive surface proteins should exist in the genome. In yeast, a large set of LTR proteins are included in flocculation (self-adhesion), a process important in biotechnology for removal of the yeast cells by sedimentation or filtration. These flo genes are of- ten truncated in deposited genomes, but it is possible that in many cases, this is due to sequencing and
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Pan-genome Search and Storage

Pan-genome Search and Storage

The de novo read assembly problem (Compeau et al., 2011) is the problem of reconstructing a genome as a sequence of nucleotides per chromosome from a set of reads only. To this end, genome assembly software use the overlaps of the reads to assemble them into larger sequences forming an assembly. This problem has been largely studied in computational biology because of its complexity and necessity. Indeed, genome analysis relies mainly on assembled genomes. Assem- bling reads that are erroneous, potentially short and with an uneven coverage that may be low or equal to zero in some regions of the sequenced genome is very challenging. Also, the sequenced genome can be difficult to assemble as it might contain repeated regions. Furthermore, finding overlaps requires to store and index the reads to assemble, a very time and memory consuming tasks as a single run of sequencing can generate up to billions of reads. Hence, the completeness and quality of an assembly depends upon all these factors as well as on the as- sembly algorithms that can create mis-assemblies. A more detailed description of the theory and practice of read assembly is given in (Simpson and Pop, 2015).
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Updated genome sequence and annotation for the full genome of Pseudomonas protegens CHA0

Updated genome sequence and annotation for the full genome of Pseudomonas protegens CHA0

between the two genome sequences. The two genomes share 5,995 genes, whereas 142 and 120 singletons were observed for our version, respectively, to the previous one, with the majority representing hypothetical proteins. Furthermore, manual annotation of all known biocontrol features and insect pathogenicity factors was performed. This version of the genome is now suited for use in comparative genomics studies to study different aspects of biocontrol and insect pathogenicity.

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Comparative genome analysis of Yersinia

Comparative genome analysis of Yersinia

Promoter sequences in the IS1331 inverted repeats might serve as additional movable promoters for the genes suffered IS1331 insertion. IS1331 is inserted in ORF181-ORF155 intergenic re[r]

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Gene family-free genome comparison

Gene family-free genome comparison

also integrate additional information such as functional similarity. Such informa- tion can be obtained from various databases, most notably, from the Gene Ontology database [9]. Family-free genome comparisons of this kind may give further insights into the functional organization of the genome. Moreover, family-free genome com- parison can also be performed based on distances between genes, instead of simi- larities. The use of distance measures could lead to the formulation of family-free distances that are suitable for distance-based phylogenetic reconstruction. A princi- pal study was performed by Martinez et al. [72], who proposed a family-free variant of the well-known double cut and join (DCJ) distance. However, their distance lacks metric properties, a not surprising consequence of the unconstrained similarity mea- sure. Even worse, phylogenetic reconstruction requires tree-additive metrics, yet the DCJ distance as well as most other gene order distances rely on the principle of parsimony, which makes them generally not tree-additive [1, 102]. Nevertheless, it is well-known that many tree reconstruction algorithms, such as the prominent neighbor-joining method [88], are to a certain extent robust against deviations from tree-additivity, which lead to the study of near-additive metrics [10]. A promising avenue of further research is to combine family-free distances with substitution rate functions of DNA substitution models. Whereas the latter provide anticipated prop- erties of tree-additivity, family-free distances could increase accuracy in reconstruc- tion, as the rate of genome rearrangements is generally much lower than the rates of point mutations. Such combined distances can be studied in a similar framework as proposed in [36] by means of affine-additive distance mappings.
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Assembly line performance and modeling

Assembly line performance and modeling

selected from the Table 2 . As per proposed methodol- ogy, next step is to estimate independent factors using Eqs. 7 , 10 and 11 . The dependent factors are to be estimated as per the Eqs. 1 , 4 , 5 and 6 . In the next step, output N is estimated using Eq. 12 . In the subsequent stage, whether the output can be further improved or not is checked. If No, the output is already optimal hence line is optimized. If Yes, lean techniques are used to improve output by reducing cycle time and time loss. This improved output is to be compared with output of other workstations. If improved output is not maximum, then output of this particular workstation still needs to be improved. Optimal solution in any vehicle assembly line can be obtained through the iterative process, wherein for a particular bottleneck station the maximum number of vehicles produced can be identified and fur- ther it can be checked whether bottleneck station can be shifted by releasing the resource constraint within the given cost constraints. This process will be repeated till the optimal solution is obtained. This proposed methodology is initially validated by using the data collected at plant A. The said methodology can also be checked whether it can be applied for other plants.
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Heteromeric assembly of P2X subunits

Heteromeric assembly of P2X subunits

Keywords: P2XR, subunit interface, homomer, heteromer, clustering, ligand binding site ASSEMBLY OF P2XRs TRIMERIC STRUCTURE OF P2XRs Early electrophysiological measurements in bullfrog sensory neu- rons and single channel analysis of HEK cell-expressed P2X2Rs predicted that there are at least three ATP molecules needed to open a P2X channel ( Bean, 1990; Ding and Sachs, 1999 ). Cross-linking studies and blue-native PAGE analysis of P2X1 and P2X3 receptors heterologously expressed in Xenopus laevis oocytes revealed the first biochemical evidence for a trimeric qua- ternary structure of P2XR channels ( Nicke et al., 1998 ). This rather unexpected architecture was subsequently confirmed by atomic force microscopy (AFM) ( Barrera et al., 2005 ), electron microscopy (EM), single particle analysis ( Mio et al., 2005; Young et al., 2008 ) and finally the first crystal structure of a P2XR, the truncated zebrafish zP2X4R ( Kawate et al., 2009 ), which consti- tuted a major breakthrough in P2XR research. Unexpectedly, the crystal structure of the acid sensing ion channel (ASIC), a mem- ber of the ENaC/DEG (epithelial sodium channels/degenerin) superfamily, which shares the same topology and was published around the same time by the Gouaux group, also revealed a trimeric structure, although the two channels show no significant amino acid sequence relationships or similarities in the folding of their extracellular domains ( Jasti et al., 2007; Gonzales et al., 2009; Kawate et al., 2009 ).
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Self-Optimization in Large Scale Assembly

Self-Optimization in Large Scale Assembly

2.3. Section assembly For the assembly of a section the left and the right side shells have to be untwisted, positioned and joined to the upper and lower shell. These four shell elements and a floor grid form a section. To fulfill tolerance requirements the biggest components of a section, the left and the right side shells, have to be positioned and untwisted. The untwisting is needed to compensate the deformation of the shell (mainly due to gravity). A robotic system has been developed for this process (Wollnack et al. (2004)) and is already used in industry. The side shells are grasped by vacuum and mechanical grippers and positioned by several linear actuators. The process is monitored by several force/torque sensors and global and local measurement systems. As the product does not have its correct shape yet, the actuators can not meet the desired grasping point exactly and also the position of measuring points are only estimations. In an iterative process the deviations between target and desired position can be determined and the residual can be minimized (Wollnack et al. (2004)). The control of the station is done automatically, as long as force limits are not exceeded. In that case a worker has to continue the process manually, as data from force/torque sensors is not considered for automatic control.
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Heteromeric assembly of P2X subunits

Heteromeric assembly of P2X subunits

Keywords: P2XR, subunit interface, homomer, heteromer, clustering, ligand binding site ASSEMBLY OF P2XRs TRIMERIC STRUCTURE OF P2XRs Early electrophysiological measurements in bullfrog sensory neu- rons and single channel analysis of HEK cell-expressed P2X2Rs predicted that there are at least three ATP molecules needed to open a P2X channel ( Bean, 1990; Ding and Sachs, 1999 ). Cross-linking studies and blue-native PAGE analysis of P2X1 and P2X3 receptors heterologously expressed in Xenopus laevis oocytes revealed the first biochemical evidence for a trimeric qua- ternary structure of P2XR channels ( Nicke et al., 1998 ). This rather unexpected architecture was subsequently confirmed by atomic force microscopy (AFM) ( Barrera et al., 2005 ), electron microscopy (EM), single particle analysis ( Mio et al., 2005; Young et al., 2008 ) and finally the first crystal structure of a P2XR, the truncated zebrafish zP2X4R ( Kawate et al., 2009 ), which consti- tuted a major breakthrough in P2XR research. Unexpectedly, the crystal structure of the acid sensing ion channel (ASIC), a mem- ber of the ENaC/DEG (epithelial sodium channels/degenerin) superfamily, which shares the same topology and was published around the same time by the Gouaux group, also revealed a trimeric structure, although the two channels show no significant amino acid sequence relationships or similarities in the folding of their extracellular domains ( Jasti et al., 2007; Gonzales et al., 2009; Kawate et al., 2009 ).
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Potato improvement by genome editing

Potato improvement by genome editing

11 th Young Scientists Meeting 2018, Braunschweig, Germany, November 14-16 Lörincz-Besenyei et al. Potato improvement by genome editing Enikö Lörincz-Besenyei 1,2 , Thorben Sprink 2 , Janina Metje 2 , Uwe Sonnewald 3 and Björn Krenz 1 1 Leibnitz Institute DSMZ-German Collection of Microorganisms and Cell Cultures, Braunschweig 2 Julius Kühn Institute, Institute for Biosafety in Plant Biotechnology, Quedlinburg

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Bioinformatic approaches for genome finishing

Bioinformatic approaches for genome finishing

First assembly algorithms date back to the times when few but relatively long Sanger reads were predominant. They are called overlap-layout-consensus approaches because they compare the reads all-against-all in order to merge overlapping parts into longer contiguous consensus sequences. The merging of reads to contigs is often done in a greedy fashion, sometimes by building an overlap graph. Some well known programs of this era are the Celera assembler [66] which was used to assemble one of the first available human genomes, ARACHNE [11], or Bambus [74]. One of the earliest assemblers that was able to cope with high throughput data, was the Newbler assembler [63, Supplem. material] that is shipped with 454 se- quencing devices. Subsequently, SSAKE [100], VCAKE [48], and SHARCGS [29] were developed to handle short reads as well. The most problematic issue of these assemblers is the computational time needed for comparing the reads all-against-all. A different class of assemblers solves this problem elegantly by using a so-called de Bruijn graph [17] for assembly [46], or more precisely a subgraph of it. The ad- vantage is that the graph can be built in time linear to the input size, as opposed to the quadratic time that the overlap-layout-consensus approaches need in general. A de Bruijn subgraph consists of nodes representing all substrings of length k, called k-mers, of the reads. The nodes are connected by an edge if two overlapping k-mers occur adjacently in one of the reads. This way, common substrings are condensed and the overlaps of the reads are collected implicitly in the graph. If the sequencing data were perfect – that is without sequencing errors and with reads longer than re- petitive regions of the genome – then the de Bruijn graph would reveal the complete genomic sequence: By following an Eulerian path that traverses every edge once, the desired complete sequence could be obtained. The de Bruijn graphs generated from real sequencing data are, however, much more complex, such that heuristics have been developed to cope with the limitations.
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First report of two complete Clostridium chauvoei genome sequences and detailed in silico genome analysis

First report of two complete Clostridium chauvoei genome sequences and detailed in silico genome analysis

lication (oriC) was comparable to that of Bacillus subtilis in structure with two regions containing DnaA boxes. Similar prophages were identi fied in the genomes of both C. chauvoei strains which also harbored hemolysin and bacterial spore formation genes. A CRISPR type I-B system with limited variations in the repeat number was identified. Sporulation and germination process related genes were homologous to that of the Clostridia clus- ter I group but novel variations for regulatory genes were identi fied indicative for strain specific control of regu- latory events. Phylogenomics showed a higher relatedness to C. septicum than to other so far sequenced genomes of species belonging to the genus Clostridium. Comparative genome analysis of three C. chauvoei circular genome sequences revealed the presence of few inversions and translocations in locally collinear blocks (LCBs). The spe- cies genome also shows a large number of genes involved in proteolysis, genes for glycosyl hydrolases and metal iron transportation genes which are presumably involved in virulence and survival in the host. Three conserved flagellar genes (fliC) were identified in each of the circular genomes. In conclusion this is the first comparative analysis of circular genomes for the species C. chauvoei, enabling insights into genome composition and virulence factor variation.
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Legal issues of digital assembly monitoring

Legal issues of digital assembly monitoring

» In the result: §§ 12a; 19 still covers the use of drones, but the  police has to mind the special aspects of drones and their. potential of intimidation[r]

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Movement‐mediated community assembly and coexistence

Movement‐mediated community assembly and coexistence

Foraging movement patterns of resource linkers and trophic linkers can have both local and regional effects by in fluencing the external environmental conditions that other organisms experience. Locally, repeated high nutrient input by resource linkers at local sites may affect the abiotic environmental fil- ter that operates during community assembly. At the one end, intense nutrient loading can cause abiotic conditions that are not tolerated well by many species. For example, ani- mal excreta enhance dissolved-oxygen depletion and ammo- nium levels in aquatic systems, which can be detrimental to fish (Wagner, 1978). At the other end, in extremely nutrient-poor systems, organic input from mobile links can decrease the strength of the environmental filter and allow greater diversity, which, for example, appears to occur for islands and surrounding shallow banks that receive nutrients through excreta from seabird colonies (Powell et al., 1991). The emergence of spatially concentrated nutrient subsidies requires particular movement behaviours such as strong localized habitat selection or defecation, for example, as per- formed by grazers that evade high temperatures by repeat- edly spending much time in the same riparian areas (Allred et al., 2013; Earl & Zollner, 2017). Additionally, unidirec- tional ‘conveyor belts’ for nutrients result from daily recur- rent movements between areas of nutrient uptake and loss, e.g. feeding and resting places (Abbas et al., 2012; Subalusky et al., 2015). Even when vector movement is less regular, aggregated resource input can arise indirectly, for example, when predator –prey spatial interactions lead to clusters of prey carcasses (Bump et al., 2009). Nutrient subsidy by mobile links also contributes to local community structuring through secondary effects. High site fidelity of aggregating meso- predatory fish attracts grazers (trophic linkers) that provide strong herbivory pressure, suppress macroalgae and thereby facilitate coral settlement and survival (Shantz et al., 2015). Note that, although local mobile-link effects can be strong enough to affect environmental filtering sensu stricto, they often create changes in external conditions that interact with biotic factors (e.g. competition effects) to shape local communities.
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Language independent transfer of assembly knowledge

Language independent transfer of assembly knowledge

and prone for translation errors. One promising attempt to cope with this challenge are language reduced or language independent instructions, as they are established for mass- or serial production. In a first attempt to create a language reduced instruction, a manual for radial compressor assembly was enriched with pictures, symbols and Blissymbols. Workers appreciated the high amount of pictures as the assembly became more visual and easier to understand [1]. When an assembly process becomes complicated, static images showed several deficits compared to dynamic ones. So called Utility-Films [2] or interactive 3D-PDFs [3] are promising attempts to cope with this challenge, while offering the opportunity to use only a limited amount of language. A survey on 40 global acting companies in 2014 shows that the usage of classic design elements like text, tables and drawings decreases and IT- based dynamic solutions like animations and videos become more and more popular, compare Fig. 1 [4].
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seq-seq-pan: building a computational pan-genome data structure on whole genome alignment

seq-seq-pan: building a computational pan-genome data structure on whole genome alignment

5. Tettelin H, Masignani V, Cieslewicz MJ, Donati C, Medini D, Ward NL, Angiuoli SV, Crabtree J, Jones AL, Durkin AS, DeBoy RT, Davidsen TM, Mora M, Scarselli M, Ros IMy, Peterson JD, Hauser CR, Sundaram JP, Nelson WC, Madupu R, Brinkac LM, Dodson RJ, Rosovitz MJ, Sullivan SA, Daugherty SC, Haft DH, Selengut J, Gwinn ML, Zhou L, Zafar N, Khouri H, Radune D, Dimitrov G, Watkins K, O’Connor KJB, Smith S, Utterback TR, White O, Rubens CE, Grandi G, Madoff LC, Kasper DL, Telford JL, Wessels MR, Rappuoli R, Fraser CM. Genome analysis of multiple pathogenic isolates of Streptococcus agalactiae: Implications for the microbial “pan-genome”. Proc Natl Acad Sci U S A. 2005;102(39):13950. 6. Beller T, Ohlebusch E. A representation of a compressed de Bruijn graph
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