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Development of the Restriction Site Tiling Analysis (RSTA) method: Accurate discovery and quantitative genotyping of genome-wide polymorphisms using nucleotide arrays

ABSTRACT: Here we developed a new high-throughput polymorphism detection and genotyping method based on identifying restriction cut site polymorphisms using a microarray platform. We compared the genomes of 20 individual urchins; 10 from the northern part of the species range (Boiler Bay, OR) and 10 from the southern part of the range (San Diego, CA). Genomic DNA from each individual was extracted, randomly sheared, then divided in half. Half was restriction digested and labeled with Cy3, half was treated as control and labeled with Cy5. Labeled DNA from a single individual was then competitively hybridized to the custom, species-specific array that had probe sequences designed to be centered on known restriction cut sites. Resulting log ratio data reflected the three possible genotypes in clusters when arrays from multiple individuals were examined at a given polymorphic locus.

ORGANISM(S): Strongylocentrotus purpuratus

SUBMITTER: Melissa Pespeni

PROVIDER: E-GEOD-20857 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Development of the Restriction Site Tiling Analysis (RSTA) method: Accurate discovery and quantitative genotyping of genome-wide polymorphisms using nucleotide arrays

Project description:Here we developed a new high-throughput polymorphism detection and genotyping method based on identifying restriction cut site polymorphisms using a microarray platform. We compared the genomes of 20 individual urchins; 10 from the northern part of the species range (Boiler Bay, OR) and 10 from the southern part of the range (San Diego, CA).

2010-03-13 | GSE20857 | GEO

ORE-seq: genome-wide absolute occupancy measurement by restriction enzyme accessibilities

Project description:Digestion with restriction enzymes is a classical approach for probing DNA accessibility in chromatin. It allows to monitor both the cut and the uncut fraction and thereby the determination of accessibility or occupancy (= 1- accessibility) in absolute terms as the percentage of cut or uncut out of the total molecules. The here presented protocol takes this classical approach to the genome-wide level. After exhaustive restriction enzyme digestion of chromatin, DNA is purified, sheared and converted into libraries for high throughput sequencing. Bioinformatic analysis counts DNA fragments cut by the restriction enzyme as well as DNA ends generated by restriction enzyme digest and derives thereof the fraction of accessible DNA. This straight forward principle is technically challenged as preparation and sequencing of the libraries leads to biased scoring of DNA fragments with ends generated by restriction enzymes versus by shearing. Our protocol includes two orthogonal approaches to correct for this bias, the “corrected cut-uncut” and the “cut-all cut” method, so that accurate measurements of absolute accessibility/occupancy at restriction sites throughout a genome are possible. The protocol is presented for the example of S. cerevisiae chromatin but may be adapted for any other species.

2023-01-01 | GSE189142 | GEO

Genome-wide survey of mRNA stability in human B-lymphoblastoid cell lines

Project description:Transcript abundance results from the balance between transcription and mRNA decay, and varies pervasively in humans. We have examined the effect of DNA variation on mRNA half-life differences by conducting a genome-wide survey of mRNA stability in seven human HapMap lymphoblastoid cell lines (LCLs). We determined the mRNA half-life for each gene from the ratio of 4-thio-uridine (4sU)-labeled nascent RNAs to total RNAs. 5,145 (46%) of 11,132 analyzed genes showed inter-individual mRNA half-life differences at a false discovery rate, FDR<0.05. As previously reported, we found transcription to be the main factor influencing transcript abundance. Although mRNA half-life explained only ~6% of transcript abundance on average, it explained ~16% for the subset of genes (~10%) showing inter-individual mRNA half-life differences (P<0.001). We confirmed previously reported correlations of mRNA half-life with transcript length, 3’-UTR length, and number of exon-junctions per kb of transcript. The number of miRNA targets in 3’-UTRs was negatively correlated with half-life (P=2.2×10-16), a new observation that is consistent with the role of miRNA in inducing mRNA degradation. Notably, coding GC and GC3 content showed positive correlations with mRNA half-life in genes with inter-individual mRNA half-life differences, implying a role of mRNA stability in shaping synonymous codon usage bias. Consistently, G or C alleles of coding SNPs were found associated with longer mRNA half-life (P=0.021). As expected, we also found that nonsense SNPs were associated with shorter mRNA half-life (P=0.009). Our results strongly suggest that inter-individual mRNA stability differences are widespread and affected by DNA sequence and composition variation.

2013-01-01 | GSE34204 | GEO

Ruler Arrays Reveal Haploid Genomic Structural Variation

Project description:A ruler array combines novel sample preparation protocols, tiling genomic microarrays, and a new computational method to turn each probe on a microarray into a ruler that measures the physical distance between defined genomic sequences regardless of the intervening sequence. The ruler array protocol involves (1) genomic DNA purification (2) digestion with a restriction enzyme (3) ligation of a biotinylated adapter molecule to the cut sites (4) purification on streptavidin beads (5) polymerase extensions from a primer complementary to the adapter using labeled dNTPs and (6) hybridization to a tiling microarray

2010-09-16 | GSE23524 | GEO

Next generation sequencing analysis of DN1 transcriptomes from Wild type, gl/gl and transgenic CD2-Ostm1 gl/gl mice

Project description:The goal of this study is to compare the transcriptome stability of DN1wt versus DN1gl/gl and DN1CD2-Ostm1gl/glTR Methods: DN1 thymocytes mRNA profiles of 19-day-old wild-type (WT), osteopetrotic grey-lethal (gl/gl) and transgenic CD2-Ostm1 gl/gl mice were generated by deep sequencing,DN1 cells from 2-3 mice per genotype were pooled, no technical replicates, using Illumina HiSeq 2000. The sequence reads that passed quality filters were analyzed at the transcript isoform level with two methods: TopHat and Cufflinks. qRTâ??PCR validation was performed using SYBR Green assays Results: DN1 gl/gl showed a distinctive transcriptome signature in comparison to DN1 wt and DN1 gl/glTR with respectivelly 146 and 205 differentially expressed genes and 205. Migration genes were significantly affected in DN1 gl/gl samples and RAC1 and S1PR1 gene expressions were confirmed using RT-qPCR. Conclusions: RNA seq allowed us to identify the enhanced expression of migration genes (RAC1 and S1PR1) in DN1gl/gl, that were both normalized in the DN1 cells from transgenic gl/glTR mice, which suggests defective T cell migration associated to the osteopetrotic thymus phenotype. DN1 thymocytes mRNA profiles of 19-day-old wild-type (WT), osteopetrotic grey-lethal (gl/gl) and transgenic CD2-Ostm1 gl/gl mice were compared by deep sequencing,DN1 cells from 2-3 mice per genotype were pooled, no technical replicates, using Illumina HiSeq 2000.

2016-09-01 | E-GEOD-72184 | biostudies-arrayexpress

Global SLAM-Seq for accurate mRNA decay determination and identification of NMD targets

Project description:Gene expression analysis requires accurate measurements of global RNA degradation rates, earlier problematic with methods disruptive to cell physiology. Recently, metabolic RNA labeling emerged as an efficient and minimally invasive technique applied in mammalian cells. Here, we have adapted SH-Linked Alkylation for the Metabolic Sequencing of RNA (SLAM-Seq) for a global mRNA stability study in yeast using 4-thiouracil pulse-chase labeling. We assign high-confidence half-life estimates for 67.5 % of expressed ORFs, and measure a median half-life of 9.4 min. For mRNAs where half-life estimates exist in the literature, their ranking order was in good agreement with previous data, indicating that SLAM-Seq efficiently classifies stable and unstable transcripts. We then leveraged our yeast protocol to identify targets of the Nonsense-mediated decay (NMD) pathway by measuring the change in RNA half-lives; instead of steady-state RNA level changes. With SLAM-Seq, we assign 580 transcripts as putative NMD targets, based on their measured half-lives in wild-type and upf3Δ mutants. We find 225 novel targets, and observe a strong agreement with previous reports of NMD targets, 61.2 % of our candidates being identified in previous studies.This indicates that SLAM-Seq is a simpler and more economic method for global quantification of mRNA half-lives. Our adaptation for yeast yielded global quantitative measures of the NMD effect on transcript half-lives, high correlation with RNA half-lives measured previously with more technically challenging protocols, and identification of novel NMD regulated transcripts that escaped prior detection.

2022-02-28 | GSE196690 | GEO

Digital Restriction Enzyme Analysis of Methylation (DREAM) of SW48 colon cancer cell line genomic DNA.

Project description:We analyzed levels of 5-methyl cytosine nnnn CCCGGG target sites by sequential restriction digest by SmaI and XmaI enzymes, ligating Illumina adaptors to the restriction fragments and reading methylation-specific signatures at the ends of restriction fragments by paired ends Illumina high throughput sequencing. Digital restriction enzyme analysis of methylation (DREAM) was performed to determine the methylation profile of SW48 colon cancer cell line genomic DNA. Genomic DNA spiked in with unmethylated, partially methylated and fully methylated standards was sequentially cut at CCCGGG sites with the methylation-sensitive enzyme SmaI (blunt ends) and its methylation-tolerant neoschizomer XmaI (5'CCGG overhangs), creating different end sequences that represented methylation status of the CCCGGG sites. These end sequences were analyzed by Illumina high throughput sequencing. Methylation status at individual CCCGGG sites across the genome was determined by counting the methylated reads with the CCGGG signature and unmethylated reads with the GGG signature at the beginnings of the sequencing reads after alignment to the human genome.

2015-09-08 | E-GEOD-66296 | biostudies-arrayexpress

Genome-wide promoter methylation analysis of human osteoarthritis

Project description:Osteoarthritis (OA) is a chronic disease of the joint characterized by a progressive degradation of articular cartilage and subchondral bone. In healthy tissue, specialized cells called chondrocytes are regulating a balanced cartilage catabolism and anabolism. By contrast osteoarthritic joints are characterized by a dramatic increase of cartilage catabolism, due to changes of gene expression patterns within chondrocytes. To identify potential epigenetic differences regulating this process a genome-wide methylation screening of paired unaffected and osteoarthritic knee cartilage samples was performed. Therefore samples of macroscopic arthritic and non-arthritic cartilage areas of the femoral condyle of five female patients were collected and DNA isolation was performed. For being able to investigate methylation changes on a genome-wide scale using only limited amounts of DNA a specific amplification protocol for mainly methylated DNA has been established, based on combinations of different methylation-sensitive and M-bM-^@M-^Sindependent restriction digestions. The amplified DNA was then labeled and hybridized onto Agilent M-bM-^@M-^\Human Promoter Whole GenomeM-bM-^@M-^] microarrays. A random variance t-test for paired (per patient) samples was performed, identifying 1214 differentially methylated genetic targets between arthritic and non-arthritic samples. The biological relevance of these genes was then further investigated via Gene Ontology (GO) and KEGG pathway analysis. DNA isolated of paired arthritic and non-arthritic knee cartilage samples of five different female osteoarthritis patients (10 samples) was methylation-specifically amplified using combinations of methylation-sensitive and -insensitive restriction enzymes. Amplicons were dye labeled (Cy3) and hybridized onto 2x244k Agilent Human Promoter microarrays.

2014-07-01 | E-GEOD-48422 | biostudies-arrayexpress

Genome-wide identification of transcriptional targets for GR in Caki-1 cells

Project description:To investigate the genome-wide transcription targets of GR in renal cell carcinoma, cleavage under targets and tagmentation (CUT&Tag) was performed in Caki-1 cells using antibodies againstGR. Following CUT&Tag, DNAs were amplified using non-biased conditions, labeled, and sequenced with Illumina NovaSeq 150PE.

2023-10-24 | GSE245752 | GEO

Genome-wide identification of transcriptional targets for H2AK119ub and H3K36me2 in LP-1 control and PSMD14 knockdown cells

Project description:To investigate the PSMD14-NSD2 complex track with H2AK119ub or H3K36me2, CUT&Tag was performed in LP-1 cells infected with lentiviruses carrying control shRNA or PSMD14 shRNA using antibodies against H2AK119ub or H3K36me2. Following CUT&Tag, DNAs were amplified using non-biased conditions, labeled, and sequenced with Illumina NovaSeq 150PE.

2023-10-29 | GSE240005 | GEO

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