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A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing

ABSTRACT: A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing

PROVIDER: PRJNA727016 | ENA |

REPOSITORIES: ENA

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			Action	DRS
	SRR14410000.fastq.gz	Fastqsanger.gz
	SRR14410001.fastq.gz	Fastqsanger.gz
	SRR14410002.fastq.gz	Fastqsanger.gz
	SRR14409966.fastq.gz	Fastqsanger.gz
	SRR14409967.fastq.gz	Fastqsanger.gz

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Project description:A genome wide microarray, covering the 10546 presently known and predicted CDS, has been constructed with the Agilent in-situ synthesized 60-mers technology. 1) A library of 10 oligonucleotides per CDS has been design by the manufacturer. 2) A computational step has been performed to determine 3' position, intron position and cross-hybridization rate of all probes. These data have been compiled to attribute a score to each probe and thus, to select a first set of 4 oligonucleotides per CDS. 3) Microarray v.2 (GPL10115), manufactured with this design, have been used for a selection procedure based on the use of transcriptomic experimental data generated with diverse culture conditions. To maximize the number of expressed CDS, experimental data were generated with a large range of growth conditions covering crucial physiological changes from key steps of vegetative growth and sexual development.For each condition, four biological replicates were done to increase the statistical power of the analysis. An oligonucleotide scoring has been settled to select the best oligonucleotide per CDS. The final array design, Microarray v.3 (GPL10116), contained 10,539 probes for nuclear CDS. The array was enriched with 17 mitochondrial CDS probes that did not undergo the experimental screening. Each probe was replicated four times on each 44K array to improve the statistical significance of results. Before using in industrious transcriptomic experiment, ultimate qualification steps have been carried out. First, DNA extracted from the/ S/ and /s /strains was hybridized with the final design Microarray v.III. The apha_ORF and /het/ genes were identified as polymorphic between /s/ and /S/ strains (|FC|>2; a/ =/0.001) confirming previous genetic and molecular analyses (Turcq B etal.,1990*,* /Current genetics, /Sellem et al., 1990, Mol. Gen. Genet.). In second qualification step, a self-to-self hybridization experiment, replicated three times, has been /per/formed with a commonreference: 89.3% of the probes displayed a signal-to-standard-deviation ratio >3. The final microarray design is referenced as AMADID 018343. Microarray slides are available to the research community from Agilent.

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A short report on optimization of nucleic acid probes by DNA microarray synthesis and next generation sequencing

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