Massive sequencing has transformed the field of genome biology due to the continuous introduction and evolution of new methods. In recent years, the technologies available to read through genomes have undergone an unprecedented rate of development in terms of cost-reduction. Generating sequence data has essentially ceased to be a bottleneck for analyzing genomes instead to be replaced by limitations in sample preparation and data analysis. In this work, new strategies are presented to increase both the throughput of library generation prior to sequencing, and the informational content of libraries to aid post-sequencing data processing. The protocols developed aim to enable new possibilities for genome research concerning project scale and sequence complexity. The first two papers that underpin this thesis deal with scaling library production by means of automation. Automated library preparation is first described for the 454 sequencing system based on a generic solid-phase polyethylene-glycol precipitation protocol for automated DNA handling. This was one of the first descriptions of automated sample handling for producing next generation sequencing libraries, and substantially improved sample throughput. Building on these results, the use of a double precipitation strategy to replace the manual agarose gel excision step for Illumina sequencing is presented. This protocol considerably improved the scalability of library construction for Illumina sequencing. The third and fourth papers present advanced strategies for library tagging in order to multiplex the information available in each library. First, a dual tagging strategy for massive sequencing is described in which two sets of tags are added to a library to trace back the origins of up to 4992 amplicons using 122 tags. The tagging strategy takes advantage of the previously automated pipeline and was used for the simultaneous sequencing of 3700 amplicons. Following that, an enzymatic protocol was developed to degrade long range PCR-amplicons and forming triple-tagged libraries containing information of sample origin, clonal origin and local positioning for the short-read sequences. Through tagging, this protocol makes it possible to analyze a longer continuous sequence region than would be possible based on the read length of the sequencing system alone. The fifth study investigates commonly used enzymes for constructing libraries for massive sequencing. We analyze restriction enzymes capable of digesting unknown sequences located some distance from their recognition sequence. Some of these enzymes have previously been extensively used for massive nucleic acid analysis. In this first high throughput study of such enzymes, we investigated their restriction specificity in terms of the distance from the recognition site and their sequence dependence. The phenomenon of slippage is characterized and shown to vary significantly between enzymes. The results obtained should favor future protocol development and enzymatic understanding. Through these papers, this work aspire to aid the development of methods for massive sequencing in terms of scale, quality and knowledge; thereby contributing to the general applicability of the new paradigm of sequencing instruments. / <p>QC 20121126</p>

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The current method to determine a mosquito specimen to a certain species is by morphological keys basically following the taxonomy developed by Carl Linnaeus in the 1700. Since Watson and Crick presented their model of the double-helix DNA in 1953, a new era of molecular based taxonomic studies have revolutionized the field. The revolution is not in terms of how the classification of species is done but how the biological diversity is seen. However, morphological, ecological and behavioral characteristics are still important and are used together with the information a gene or whole genome can give. DNA barcoding is one of the promising methods for molecular identification. A small segment of a gene, approximately 400-1000 base pairs (bp), are examined by a Polymerase chain reaction (PCR) and sequencing. Like the barcodes in the grocery store these sequences work like unique ID: s for every species. This thesis shows how a fast DNA extraction method could be combined with DNA barcoding to get a 658-bp segment of the mitochondrial gene cytochrome c oxidase subunit 1 (COI) from different species of the mosquito family Culicidae. A total of 15 thoraxes or wings, from individual specimen of mosquitoes, were examined and 11 different barcode sequences could be retrieved. Six correspond to already published COI sequences and could therefore be determined to the species level, including a sequence from a new species for Sweden, Aedes (Ochlerotatus) nigrinus. All mosquitoes were collected during the national inventory of species in summer of 2012 in Sweden, ”Myggjakten”, and have been morphological examined by experts at the National Veterinary Institute (SVA) prior to molecular determination. This thesis also highlights the importance of building a reference library of barcode sequences, so DNA barcoding could become an effective diagnostic tool. Inventory projects like “Myggjakten” may, if repeated, provide excellent material for such a library collection of barcode data. / När en stickmyggsart skall artbestämmas är den vanligaste metoden att använda morfologiska nycklar. I princip görs det här efter den taxonomi som Carl von Linne utvecklade på 1700-talet. Men sedan Watson och Crick presenterade sin DNA modell 1953 så har dock en ny era av molykylärt baserade metoder revolutionerat taxonomin. Förändringen består egentligen inte i hur vi klassificerar och använder taxonomin utan mer hur vi ser på den biologiska mångfalden. Morfologiska och ekologiska studier, samt studier av arters beteende, är fortfarande viktiga och komplementerar den molekylära informationen från ett genom eller från en enstaka gen. DNA barcoding är en av de lovande nya molekylära metoderna för artbestämning. Ett litet segment av en gen, på ungefär 400-1000 baspar (bp), undersöks med hjälp av polymeras-kedjereaktion (PCR) och sekvensering. Likt streckkoder i livsmedelsbutiken ger metoden ett unikt ID för varje art. Den här studien visar hur en snabb DNA-extraktionsmetod kan kombineras med DNA barcoding, för att ge en 658-bp lång DNA-sekvens, från den mitokondriella genen cytokrom c oxidas subunit 1 (COI) från olika arter av myggfamiljen Culicidae. I undersökningen ingick 15 mellankroppar eller vingar från individuella stickmyggor och av dessa kunde 11 olika barcode sekvenser utläsas. Sex av dessa stämde överrens med redan publicerade COI-sekvenser och kunde bestämmas till artnivå, varav en av sekvenserna kommer från den nyligen i Sverige funna morfologiskt artbestämda Aedes (Ochlerotatus) nigrinus. Stickmyggorna i detta arbete insamlades av privatpersoner på olika ställen i Sverige under sommaren 2012 i det nationella mygginsamlingsprojektet ”Myggjakten”. Dessa artbestämdes morfologiskt av personal på Statens veterinärmedicinska anstalt (SVA) innan de artbestämdes molekylärt. Det här arbetet belyser även vikten av att bygga upp ett referensbibliotek av barcode sekvenser för att DNA-barcoding ska kunna bli ett effektivt diagnostiskt verktyg vid studier av vektorburna zoonoser. Nationella projekt som Myggjakten kan vara mycket användbara för insamling av sådana data.

The Myristicaceae, or nutmeg family, consists of 21 genera and about 500 species of dioecious canopy to sub canopy trees that are distributed worldwide in tropical rainforests. The Myristicaceae are of considerable ecological and ethnobotanical significance as they are important food for many animals and are harvested by humans for timber, spices, dart/arrow poison, medicine, and a hallucinogenic snuff employed in medico-religious ceremonies. Despite the importance of the Myristicaceae throughout the wet tropics, our taxonomic knowledge of these trees is primarily based on the last revision of the five neotropical genera completed in 1937. The objective of this thesis was to perform a molecular and morphological study of the neotropical genera Compsoneura and Virola. To this end, I generated phylogenetic hypotheses, surveyed morphological and genetic diversity of focal species, and tested the ability of DNA barcodes to distinguish species of wild nutmegs. Morphological and molecular analyses of Compsoneura. indicate a deep divergence between two monophyletic clades corresponding to informal sections Hadrocarpa and Compsoneura. Although 23 loci were tested for DNA variability, only the trnH-psbA intergenic spacer contained enough variation to delimit 11 of 13 species sequenced. A morphological and molecular investigation of Compsoneura capitellata showed little discrete morphological variation among populations but significant genetic structure among populations. Phylogenetic analysis of Virola also revealed a deep molecular divergence between two clades having numerous contrasting morphologies. In contrast to Compsoneura, the trnH-psbA intergenic spacer failed to differentiate the majority of Virola species tested. An infraspecific morphological and molecular study of V. sebifera and V. loretensis showed that each of these species contains morphologically and ecologically discrete sympatric morphotypes that likely represent new species. In total, this investigation found 5 provisional new species from fewer than 600 collections at biological stations in Ecuador and Peru where these new species were among the most abundant trees in the forest. This suggests that much diversity likely remains to be described in the Myristicaceae and other tropical plant families.

Background: The automotive industry accounts for a large part of the European economic structure. Due to both economical and environmental impacts, the industry has undergone substantial changes and companies have to increase their efficiency to stay competitive. An improvement-area, which can be directly influenced by the company is the internal material handling. A new technology that potentially supports the internal material handling process is the radio frequency identification (RFID) technology, which is perceived as a fruitful successor of the common barcoding technology. Even though the RFID technology shows multiple benefits over the barcoding technology, many companies are still reluctant to the application of the new method. The authors therefore strive to provide a deeper understanding of the following two research questions:   RQ 1: To what extent and how is RFID currently applied in the internal material handling process in the investigated automotive companies? RQ 2: For what reasons did the investigated automotive companies decide to apply or not apply RFID technologies to support their internal material handling process?   Purpose: The purpose of this thesis is to show through a multiple case study to what extent and how RFID technology is currently applied to support the internal material handling process in a number of companies in the automotive sector, both original equipment manufacturers (OEMs) and suppliers. Thereupon the main reasons for or against the application of RFID in these companies are examined.   Method: This thesis adopts a positivistic perspective and a deductive approach. It is designed as a qualitative multiple case study carried out in four different companies with five different plants in the automotive industry. Empirical data was gathered through interviews. The analysis is based on primary as well as secondary data.   Conclusions: Throughout the course of the study it became apparent that the RFID technology is on the radar of all investigated companies. Only Scania Zwolle, Volvo Skövde and Bosch Homburg apply the technology and see concrete benefits in the usage of RFID above barcoding. The extent of application here differs from a large scale to a small scale. The three companies name benefits such as an improved automatic tracking &amp; tracing system with improved real-time data quality and a reduction in costs, which is mainly achieved through a reduction of manual labour. Additionally they face benefits, which are business-specific such as the possibility for automatic alerts throughout the internal material handling process at Scania Zwolle, the need for a ‘silent’ successor over barcoding at Volvo Skövde and a supporting tool for their lean management program at Bosch Homburg. VDL Nedcar Born and Scania Oskarshamn in turn name concrete reasons for not applying the technology. VDL Nedcar Born is undergoing substantial changes in their production facility which currently has priority and Scania Oskarshamn does not see benefits that outweigh the high costs for the RFID technology.

Biodiversity across the globe is threatened. Rapid surveying and monitoring techniques are required to understand the origin of the threats to biodiversity and to enable conservation actions to be undertaken. Kuwait is an arid desert country with a small flora of only 402 species. This flora is endangered by environmental factors, overgrazing, and human activities. DNA barcoding the flora and using Next Generation Sequencing (NGS) technologies allowed us to identify plants to species level, conduct a molecular taxonomic revision, and distinguish plant diversity found in soil environmental DNA samples. After investigating the discriminatory power of five commonly used DNA markers from plastid (matK, rbcL, trnH-psbA, trnL) and a nuclear genome (ITS2) on four largest genera of the flora using phylogenetics reconstruction tree based methods, two barcoding markers (rbcL and ITS2) were assigned to build a DNA reference library of the flora. Furthermore, the DNA reference library was tested to identify the plant diversity found below-ground level and comparing it with that above-ground, using environmental soil samples collected from both species rich and poor habitats in Kuwait by applying high-throughput sequencing methods. The DNA database provided in this study could be used as a reference library for the identification process and contribute towards the future of molecular taxonomy, biodiversity and ecological research in Kuwait.

Tropical rain forests around the world suffer from deforestation, which is caused mainly by small-scale farmers. These farmers largely employ slash-and-burn methods to clear the land for agricultural settlement. Agroforestry systems are widely found in the humid tropics, where they could have great potential to increase the productivity of farming systems and sustain continuous crop production and they are also supposed to conserve biodiversity. As a group of freshwater invertebrates, dragonflies (Odonata) are commonly used as ecological indicators of freshwater ecosystems. The main objective of the study is to assess the impact of land use changes on dragonflies (Odonata) species richness and diversity, namely primary and secondary forest, cocoa agroforest and slash-and-burn agriculture in the Tropical Africa. We hypothesize, that the species richness and diversity of dragonflies decrease with disturbance of the ecosystems, along the land-use changes gradient.

Desert environments are characterised by harsh conditions and possess low biodiversity largely caused by abiotic factors such as; low precipitation, soil organic matter, high temperatures, high levels of evapo-transpiration, pH and salinity. These factors significantly reduce primary production, which influences the availability of food resources for deserts organisms. The diversity and the drivers of diversity for below ground invertebrates including Collembola (springtails) are relatively unknown in the Namib Desert. Previous morphological studies have found only five species on the basis of traditional taxonomy. This study assesses the diversity of Namib Desert Collembola and determines the effect of environmental parameters on this diversity, The diversity of Namib Desert Collembola was assessed using DNA Barcoding. The sequence information of the 178 Collembola specimens, taken from mitochondrial barcoding using the Cytochrome-c oxidase subunit I (COI) gene, was analyzed and Molecular Operational Taxonomic Units (MOTUs) were defined. Collembola community responses to soil physicochemical properties were investigated by using Redundancy Analysis (RDA). MOTUs were successfully indentified to family level (Isotomidae, Neanuridae and Sminthuridae). The researcher found a total of 30 MOTUs, most of which showed limited geographical localisation. The mtDNA COI (barcode) locus revealed high levels of previously unreported genetic diversity of Collembola in the Namib Desert. The RDA indicated that none of the soil physicochemical properties significantly drove variation in Collembola community composition. However, total soil nitrogen was shown to be a strong but not significant driver of variation in community composition (p<0,054). The taxonomic identification of the Collembola specimens was also attempted using traditional morphological analysis. A total of 23 individuals, collected from pitfall traps or extracted from soil samples, were selected for identification. Available European keys were used for identification to genus level where possible. A total eight of specimens were identified to genus level (Folsomides sp), 14 to family level (Entomobryidae) and one to order level (Symphypleona). Both Symphypleona and Entomobryidae were previously unreported from the Namib Desert. The Folsomides genus and the family Entomobryidae were the most abundant groups. This research suggests that soil dwelling Collembola in the Namib Desert have a much higher level of diversity than previously known. However, the study also highlighted the need for a more comprehensive database for Namib Collembola that includes COI sequence data as well as the morphological identification of species. / Dissertation (MSc)--University of Pretoria, 2018. / National Research Foundation (NRF) / Genetics / MSc / Unrestricted

The cosmopolitan echiniscid genus Hypechiniscus contains exclusively rare species. In this contribution, by combining statistical morphometry and molecular phylogeny, we present qualitative and quantitative aspects of Hypechiniscus diversity, which remained hidden under the two purportedly cosmopolitan species: H. gladiator and H. exarmatus. A neotype is designated for H. gladiator from Creag Meagaidh (Scotland), and an informal re-description is provided for H. exarmatus based on animals from Creag Meagaidh and the Isle of Skye (Inner Hebrides). Subspecies/forms of H. gladiator are suppressed due to the high developmental variability of the cirrus dorsalis. At the same time, four species of the genus are described: H. daedalus sp. nov. from Roan Mountain and the Great Smoky Mountains (Southern Appalachian Mountains, USA), H. flavus sp. nov. and H. geminus sp. nov. from the Yatsugatake Mountains (Honshu, Japan), and H. cataractus sp. nov. from the Malay Archipelago (Borneo and the Moluccas). Dorsal and ventral sculpturing, together with morphometric traits, are shown to be the key characters that allow for the phenotypic discrimination of species within the genus. Furthermore, the morphology of Hypechiniscus is discussed and compared to that of the most similar genera, Pseudechiniscus and Stellariscus. Finally, a diagnostic key to all recognized Hypechiniscus species is provided.

Between 33 and 91 percent of marine species are currently undescribed, with the majority occurring in tropical and offshore environments. Sponges act as important microhabitats and promote biodiversity by harboring a wide variety of macrofauna and microbiota, but little is known about the relationships between the sponges and their symbionts. This study uses DNA barcoding to examine the macrofaunal communities associated with sponges of the central Saudi Arabian Red Sea, a drastically understudied ecosystem with high biodiversity and endemism. In total, 185 epifaunal and infaunal operational taxonomic units (OTUs) were distinguished from the 1399 successfully-sequenced macrofauna individuals from 129 sponges representing seven sponge species, one of which (Stylissa carteri) was intensively studied. A significant difference was found in the macrofaunal community composition of Stylissa carteri along a cross-shelf gradient using relative OTU abundance (Bray-Curtis diversity index). The abundance of S. carteri also follows a cross-shelf gradient, increasing with proximity to shore. The difference in macrofaunal communities of several species of sponges at one location was found to be significant as well, using OTU presence (binary Jaccard diversity index). Four of the seven sponge species collected were dominated by a single annelid OTU, each unique to one sponge species. A fifth was dominated by four arthropod OTUs, all species-specific as well. Region-based diversity differences may be attributed to environmental factors such as reef morphology, water flow, and sedimentation, whereas species-based differences may be caused by sponge morphology, microbial abundances, and chemical defenses. As climate change and ocean acidification continue to modify coral reef ecosystems, understanding the ecology of sponges and their role as microhabitats may become more important. This thesis also includes a supplemental document in the form of a spreadsheet showing the number of macrofauna individuals of each OTU found within each sponge sample.