Increased Throughput by Parallelization of Library Preparation for Massive Sequencing

Background: Massively parallel sequencing systems continue to improve on data output, while leaving labor-intensive library preparations a potential bottleneck. Efforts are currently under way to relieve the crucial and time-consuming work to prepare DNA for high-throughput sequencing. Methodology/Principal Findings: In this study, we demonstrate an automated parallel library preparation protocol using generic carboxylic acid-coated superparamagnetic beads and polyethylene glycol precipitation as a reproducible and flexible method for DNA fragment length separation. With this approach the library preparation for DNA sequencing can easily be adjusted to a desired fragment length. The automated protocol, here demonstrated using the GS FLX Titanium instrument, was compared to the standard manual library preparation, showing higher yield, throughput and great reproducibility. In addition, 12 libraries were prepared and uniquely tagged in parallel, and the distribution of sequence reads between these indexed samples could be improved using quantitative PCR-assisted pooling. Conclusions/Significance: We present a novel automated procedure that makes it possible to prepare 36 indexed libraries per person and day, which can be increased to up to 96 libraries processed simultaneously. The yield, speed and robust performance of the protocol constitute a substantial improvement to present manual methods, without the need of extensive equipment investments. The described procedure enables a considerable efficiency increase for small to midsiz

Accuracy and quality assessment of 454 GS-FLX Titanium pyrosequencing

<p>Abstract</p> <p>Background</p> <p>The rapid evolution of 454 GS-FLX sequencing technology has not been accompanied by a reassessment of the quality and accuracy of the sequences obtained. Current strategies for decision-making and error-correction are based on an initial analysis by Huse <it>et al. </it>in 2007, for the older GS20 system based on experimental sequences. We analyze here the quality of 454 sequencing data and identify factors playing a role in sequencing error, through the use of an extensive dataset for Roche control DNA fragments.</p> <p>Results</p> <p>We obtained a mean error rate for 454 sequences of 1.07%. More importantly, the error rate is not randomly distributed; it occasionally rose to more than 50% in certain positions, and its distribution was linked to several experimental variables. The main factors related to error are the presence of homopolymers, position in the sequence, size of the sequence and spatial localization in PT plates for insertion and deletion errors. These factors can be described by considering seven variables. No single variable can account for the error rate distribution, but most of the variation is explained by the combination of all seven variables.</p> <p>Conclusions</p> <p>The pattern identified here calls for the use of internal controls and error-correcting base callers, to correct for errors, when available (e.g. when sequencing amplicons). For shotgun libraries, the use of both sequencing primers and deep coverage, combined with the use of random sequencing primer sites should partly compensate for even high error rates, although it may prove more difficult than previous thought to distinguish between low-frequency alleles and errors.</p

Skolans matematik : En kritisk analys av den svenska skolmatematikens förhistoria, uppkomst och utveckling

I argue that common beliefs regarding mathematics originate in the practices of elementary mathematics instruction rather than in science. While learning how to solve mathematical problems in school, we come to believe that mathematics has a set of properties in itself, for instance that it is useful in everyday life, even though this is not necessarily so. I call this object of belief the mathematics of schooling (skolans matematik), while the system of practices by which the belief is produced is called mathematics education (skolmatematik). I introduce a terminology inspired by psychoanalytic theory to describe the peculiar properties of the mathematics of schooling and suggest that it can be understood as a sublime object of an ideology propagated by the system of education. To substantiate this claim I give an overview of the history of Swedish mathematics education, based on curricula, textbooks, discussions in teacher magazines, and other published material – covering in general terms the eighteenth to the twenty-first century. The historical narrative moves between social factors determining the practices of mathematics education, the changing ideas about mathematics expressed in these contexts, and the interplay between external social factors and internal “ideological” meaning. My conclusion is that while elementary arithmetics is, and should be, a part of common knowledge, the mathematics of schooling is something quite different. This object is thoroughly ideological and plays a central part in society mainly by making the social effects of mathematics education – keeping children away from production while sorting them – to appear as something else, namely as most often failed attempts to give children a necessary knowledge of mathematics

Methods to Prepare DNA for Efficient Massive Sequencing

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.QC 20121126</p

How mathematics education became a ritual

The following essay, which is based on the first half of my talk at the GDM conference in Potsdam 2017, should be read as a conjecture concerning mathematics education, intending to spur discussion and open up new pathways for research. I start by trying to show that present day mathematics education can be interpreted as a ritual, after which I indicate five distinct origins of this ritual. The reason why the second half of my talk is not included in this essay, is that an account of the explanatory framework presented there can be found in texts published elsewhere (Lundin, 2013; Christensen & Lundin, 2017)

Programmering f\uf6r en problembaserad undervisning i gymnasieskolan

I det h\ue4r kapitlet beskrivs ett fortbildningsprojekt vars syfte var att \uf6ka kunskapen om vilka m\uf6jligheter till l\ue4rande i matematik som programmering kan erbjuda i gymnasieskolan. I projektet fick gymnasieelever tillsammans med l\ue4rarstudenter och gymnasiel\ue4rare \ue4gna sig \ue5t probleml\uf6sning med hj\ue4lp av programmering. Speciellt f\uf6r detta projekt var att programmeringen gjordes i Mathematica, vars programmeringsspr\ue5k skiljer sig ganska mycket fr\ue5n de som vanligen anv\ue4nds i grundskolan. F\uf6rfattarna menar att fokus vid programmering i matematikundervisning alltf\uf6r ofta ligger p\ue5 algoritmer, och argumenterar f\uf6r att man i st\ue4llet b\uf6r fokusera p\ue5 programmering som skapande, d\ue4r fr\ue5gorna snarare handlar om vad som ska skapas, vad det skapade ska gestalta, och hur det b\uf6r vara strukturerat