Search results for "DNA sequencing"
showing 10 items of 237 documents
Galaxy LIMS for next-generation sequencing.
2013
Abstract Summary: We have developed a laboratory information management system (LIMS) for a next-generation sequencing (NGS) laboratory within the existing Galaxy platform. The system provides lab technicians standard and customizable sample information forms, barcoded submission forms, tracking of input sample quality, multiplex-capable automatic flow cell design and automatically generated sample sheets to aid physical flow cell preparation. In addition, the platform provides the researcher with a user-friendly interface to create a request, submit accompanying samples, upload sample quality measurements and access to the sequencing results. As the LIMS is within the Galaxy platform, the …
Adaptive reference-free compression of sequence quality scores
2014
Motivation: Rapid technological progress in DNA sequencing has stimulated interest in compressing the vast datasets that are now routinely produced. Relatively little attention has been paid to compressing the quality scores that are assigned to each sequence, even though these scores may be harder to compress than the sequences themselves. By aggregating a set of reads into a compressed index, we find that the majority of bases can be predicted from the sequence of bases that are adjacent to them and hence are likely to be less informative for variant calling or other applications. The quality scores for such bases are aggressively compressed, leaving a relatively small number at full reso…
ChIP-Seq from Limited Starting Material of K562 Cells and Drosophila Neuroblasts Using Tagmentation Assisted Fragmentation Approach
2019
Chromatin immunoprecipitation is extensively used to investigate the epigenetic profile and transcription factor binding sites in the genome. However, when the starting material is limited, the conventional ChIP-Seq approach cannot be implemented. This protocol describes a method that can be used to generate the chromatin profiles from as low as 100 human or 1,000 Drosophila cells. The method employs tagmentation to fragment the chromatin with concomitant addition of sequencing adaptors. The method generates datasets with high signal to noise ratio and can be subjected to standard tools for ChIP-Seq analysis.
Molecular study of porcine transmissible gastroenteritis virus after serial animal passages revealed point mutations in S protein
2010
Porcine respiratory coronavirus is related genetically to porcine transmissible gastroenteritis virus with a large deletion in S protein. The respiratory virus is a mutated form that may be a consequence of the gastroen- teritis virus's evolution. Intensive passages of the virus in its natural host may enhance the appearance of mutations and therefore may contribute to any attenuated form of the virus. The objective of this study was to characterize the porcine transmissible gastroenteritis virus TMK22 strain after passages in piglets from 1992 until 2007. A typical experimental infection, molecular characterization, and serological analysis were also carried out to further char- acterize a…
Phylogeny of Veronica in the Southern and Northern Hemispheres based on plastid, nuclear ribosomal and nuclear low-copy DNA
2010
The cosmopolitan and ecologically diverse genus Veronica with approximately 450 species is the largest genus of the newly circumscribed Plantaginaceae. Previous analyses of Veronica DNA sequences were in stark contrast to traditional systematics. However, analyses did not allow many inferences regarding the relationship between major groups identified, hindering further analysis of diversification and evolutionary trends in the genus. To resolve the backbone relationships of Veronica, we added sequences from additional plastid DNA regions to existing data and analyzed matching data sets for 78 taxa and more than 5000 aligned characters from nuclear ribosomal DNA and plastid DNA regions. The…
Unveiling distribution patterns of freshwater phytoplankton by a next generation sequencing based approach.
2012
The recognition and discrimination of phytoplankton species is one of the foundations of freshwater biodiversity research and environmental monitoring. This step is frequently a bottleneck in the analytical chain from sampling to data analysis and subsequent environmental status evaluation. Here we present phytoplankton diversity data from 49 lakes including three seasonal surveys assessed by next generation sequencing (NGS) of 16S ribosomal RNA chloroplast and cyanobacterial gene amplicons and also compare part of these datasets with identification based on morphology. Direct comparison of NGS to microscopic data from three time-series showed that NGS was able to capture the seasonality in…
Measuring RNA polymerase activity genome-wide with high-resolution run-on-based methods
2019
The biogenesis of RNAs is a multi-layered and highly regulated process that involves a diverse set of players acting in an orchestrated manner throughout the transcription cycle. Transcription initiation, elongation and termination factors act on RNA polymerases to modulate their movement along the DNA template in a very precise manner, more complex than previously anticipated. Genome-scale run-on-based methodologies have been developed to study in detail the position of transcriptionally-engaged RNA polymerases. Genomic run-on (GRO), and its many variants and refinements made over the years, are helping the community to address an increasing amount of scientific questions, spanning an incr…
A method for genome-wide analysis of DNA helical tension by means of psoralen-DNA photobinding
2010
The helical tension of chromosomal DNA is one of the epigenetic landmarks most difficult to examine experimentally. The occurrence of DNA crosslinks mediated by psoralen photobinding (PB) stands as the only suitable probe for assessing this problem. PB is affected by chromatin structure when is done to saturation; but it is mainly determined by DNA helical tension when it is done to very low hit conditions. Hence, we developed a method for genome-wide analysis of DNA helical tension based on PB. We adjusted in vitro PB conditions that discern DNA helical tension and applied them to Saccharomyces cerevisiae cells. We selected the in vivo cross-linked DNA sequences and identified them on DNA …
Immunological Methods for Analysis of Recombinant Proteins
1998
We want to introduce researchers to techniques that help to solve some problems in the work of the molecular biologist. After transformation of recombinant DNA in E. coli cells many clones are usually produced, and the same situation appears if recombinant DNA expression libraries are available. Furthermore, if appropriate monoclonal or polyclonal antibodies are available, the method of immunoscreening of colonies for direct immunological detection of translational products of cloned genes can be used. Selected clones could be chosen for further studies such as determination of their primary structure by DNA sequencing and for characterization of an appropriate expressed protein.
TAF-ChIP: An ultra-low input approach for genome wide chromatin immunoprecipitation assay
2018
Chromatin immunoprecipitation (ChIP) followed by next generation sequencing is an invaluable and powerful technique to understand transcriptional regulation. However, ChIP is currently limited by the requirement of large amount of starting material. This renders studying rare cell populations very challenging, or even impossible. Here, we present a tagmentation-assisted fragmentation ChIP (TAF-ChIP) and sequencing method to generate high-quality datasets from low cell numbers. The method relies on Tn5 transposon activity to fragment the chromatin that is immunoprecipitated, thus circumventing the need for sonication or MNAse digestion to fragment. Furthermore, Tn5 adds the sequencing adapto…