Design and experimental evaluation of a minimal, innocuous watermarking strategy to distinguish near-identical DNA and RNA sequences
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ABSTRACT:
PROVIDER: PRJNA558994 | ENA |
REPOSITORIES: ENA
Dataset's files
| Action | DRS | |||
|---|---|---|---|---|
| SRR9913280_1.fastq.gz | Fastqsanger.gz | |||
| SRR9913280_2.fastq.gz | Fastqsanger.gz | |||
| SRR9913281_1.fastq.gz | Fastqsanger.gz | |||
| SRR9913281_2.fastq.gz | Fastqsanger.gz | |||
| SRR9913282_1.fastq.gz | Fastqsanger.gz |
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Project description:The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavours, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The thirteen genes encoding proteins involved in the major pathway for sugar utilization (i.e glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not alter transcript abundance, protein activity and yeast physiology. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.
2020-06-29 | GSE135470 | GEO
Project description:The construction of powerful cell factories requires intensive and extensive remodelling of microbial genomes. Considering the rapidly increasing number of these synthetic biology endeavors, there is an increasing need for DNA watermarking strategies that enable the discrimination between synthetic and native gene copies. While it is well documented that codon usage can affect translation, and most likely mRNA stability in eukaryotes, remarkably few quantitative studies explore the impact of watermarking on transcription, protein expression, and physiology in the popular model and industrial yeast Saccharomyces cerevisiae. The present study, using S. cerevisiae as eukaryotic paradigm, designed, implemented, and experimentally validated a systematic strategy to watermark DNA with minimal alteration of yeast physiology. The 13 genes encoding proteins involved in the major pathway for sugar utilization (i.e., glycolysis and alcoholic fermentation) were simultaneously watermarked in a yeast strain using the previously published pathway swapping strategy. Carefully swapping codons of these naturally codon optimized, highly expressed genes, did not affect yeast physiology and did not alter transcript abundance, protein abundance, and protein activity besides a mild effect on Gpm1. The markerQuant bioinformatics method could reliably discriminate native from watermarked genes and transcripts. Furthermore, presence of watermarks enabled selective CRISPR/Cas genome editing, specifically targeting the native gene copy while leaving the synthetic, watermarked variant intact. This study offers a validated strategy to simply watermark genes in S. cerevisiae.
| S-EPMC7309318 | biostudies-literature
Project description:F. Boonekamp et al., (2019) describe an innocuous watermarking strategy for coding regions that enables the discrimination of DNA and mRNA by sequencing through a k-mer approach and facilitates selective editing of watermarked and non-watermarked sequences. Shot-gun proteomics (LFQ) was performed on the parent and the engineered yeast strain. *Corresponding author: Pascale Daran-Lapujade (p.a.s.daran-lapujade@tudelft.nl). Department of Biotechnology, Delft University of Technology, van der Maasweg 9, 2629HZ Delft, The Netherlands
2020-09-21 | PXD016914 | Pride
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Project description:Transcriptomes distinguish human FCD subtypes
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Project description:How to design experiments that accelerate knowledge discovery on complex biological landscapes remains a tantalizing question. Here, we present OPEX, an optimal experimental design method to identify informative omics experiments for both experimental space exploration and model training. OPEX-guided exploration of Escherichia coli's cross-behavior potential, when exposed to novel biocide and antibiotic combinations, led to accelerated knowledge discovery with predictive models that are more accurate while needing 44% fewer data to train. Selecting experiments favoring broader exploration followed by fine-tuning emerged as the optimal strategy. This led to the discovery of 29 cross-protection and 4 cross-vulnerability conditions, with further validation revealing the central role of chaperones, stress response proteins and transport pumps in cross-stress exposure. This work demonstrates how active learning can be used to automate omics data collection for training accurate predictive models, evidence-driven decision making and accelerated knowledge discovery in life sciences.
2020-07-09 | GSE144604 | GEO
Project description:Genome wide DNA methylation profiling of blood samples from eight female identical twins of Han Chinese for forensic age prediction, age 21 to 32. The Illumina Infinium HumanMethylation450 BeadChip was used to obtain DNA methylation profiles across approximately 485,000 CpGs at a single-nucleotide resolution. Samples included 8 pairs of identical female twins of Han Chinese.
2015-12-08 | GSE65638 | GEO
Project description:Accelerated knowledge discovery from omics data by optimal experimental design
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Project description:BACKGROUND: Microarray comparative genomic hybridization (CGH) is currently one of the most powerful techniques to measure DNA copy number in large genomes. In humans, microarray CGH is widely used to assess copy number variants in healthy individuals and copy number aberrations associated with various diseases, syndromes and disease susceptibility. In model organisms such as Caenorhabditis elegans (C. elegans) the technique has been applied to detect mutations, primarily deletions, in strains of interest. Although various constraints on oligonucleotide properties have been suggested to minimize non-specific hybridization and improve the data quality, there have been few experimental validations for CGH experiments. For genomic regions where strict design filters would limit the coverage it would also be useful to quantify the expected loss in data quality associated with relaxed design criteria. RESULTS: We have quantified the effects of filtering various oligonucleotide properties by measuring the resolving power for detecting deletions in the human and C. elegans genomes using NimbleGen microarrays. Approximately twice as many oligonucleotides are typically required to be affected by a deletion in human DNA samples in order to achieve the same statistical confidence as one would observe for a deletion in C. elegans. Surprisingly, the ability to detect deletions strongly depends on the oligonucleotide 15-mer count, which is defined as the sum of the genomic frequency of all the constituent 15-mers within the oligonucleotide. A similarity level above 80% to non-target sequences over the length of the probe produces significant cross-hybridization. We recommend the use of a fairly large melting temperature window of up to 10 C, the elimination of repeat sequences, the elimination of homopolymers longer than 5 nucleotides, and a threshold of -1 kcal/mol on the oligonucleotide self-folding energy. We observed very little difference in data quality when varying the oligonucleotide length between 50 and 70, and even when using an isothermal design strategy. CONCLUSIONS: We have determined experimentally the effects of varying several key oligonucleotide microarray design criteria for detection of deletions in C. elegans and humans with NimbleGen's CGH technology. Our oligonucleotide design recommendations should be applicable for CGH analysis in most species.
2008-07-26 | GSE12208 | GEO