A SureSelect Custom Target Enrichment Library covering the 14 CRC risk loci, represented by 18 tagSNPs, was designed using eArray software (Agilent, Santa Clara, CA, USA). Biotinylated RNA baits were generated to capture genomic sequence within LD blocks to which tagSNPs associations mapped (Supplementary Table 2 ). LD data were extracted using SNAP46 (link) based on CEU HapMap47 (link) phase 3, imposing parameters r2>0.2 and a minor allele frequency of >2%. LD measures for tagSNPs not included in HapMap were extracted from the thousand genomes (1000g) pilot data48 (link). The region to be enriched encompassing the 8q24 risk locus was extended to 1.118 Mb centring on rs6983267. The total enrichment target of 4.683 Mb was submitted to Agilent eArray software, generating 43,380 120-mer RNA baits designed to tile the non-repetitive fraction of the test regions at 3 × coverage. Our design scale, tiling the total regions of interest after masking repeats, fitted the size ranges of commercially available Agilent SureSelect Custom Target Enrichment kits. Notably, designing baits for sequences >500–1,500 bp (depending on NGS fragment distribution) from a HindIII restriction site does not yield in improvement of the enrichment efficacy, which should be considered when facing limitations in bait numbers.
Earray software
Manufactured by Agilent Technologies
Sourced in United Kingdom, United States
EArray software is a bioinformatics tool designed for the analysis of microarray data. It provides a platform for importing, processing, and visualizing microarray data from various platforms. The software includes functionalities for normalization, statistical analysis, and the identification of differentially expressed genes.
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Lab products found in correlation
One color microarray based gene expression analysis v6.5, by Agilent Technologies (2 mentions)
Sureprint oligo technology, by Agilent Technologies (1 mentions)
Cytogenomics software, by Agilent Technologies (1 mentions)
Sureselect protocol, by Agilent Technologies (1 mentions)
Hiseq 2500 system, by Illumina (1 mentions)
Feature extraction software, by Agilent Technologies (1 mentions)
20 protocols using earray software
1
Customized Enrichment Library Design
2
Screening MLH1 Epimutation Structural Alterations
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A screen for the presence of structural alterations was performed on PBL DNA from MLH1 epimutation carriers using a custom high-definition CGH array designed with eArray Software (Agilent Technologies) and manufactured by Agilent’s SurePrint oligo technology. This comprised 15,000 probes encompassing the MLH1 locus (region Chr3:36,450,000–37,900,000 within cytoband 3p22.2), with an average probe spacing of 100 bp intervals. Bioinformatics analysis was performed in R using the 2.15.12 Bioconductor statistical packages. Results were visualized on CytoGenomics Software (Agilent Technologies).
3
Comprehensive C. albicans Genome Coverage
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Nonoverlapping head-to-tail 120-nucleotide probes were designed using the eArray software (Agilent Technologies, Santa Clara, CA). A total of 55,342 bait probes were designed to cover 6,094 C. albicans ORFs (assembly 21 SC5314; 16-Mb haploid genome; 6,218 ORFs). The first 250 nucleotides of each gene were not covered in the bait design, resulting in an average of 9 probes for each ORF. Using Megablast (v2.2.26) (76 (link)), it was verified that all genes of C. albicans were matched by at least one probe and that only a negligible fraction of the probes could be mapped on the mouse and human cDNA sequences from Ensembl and on the available G. mellonella expressed sequence tags (ESTs) and cDNA sequences from NCBI.
4
Exon-Level Microarray for Genomic Aberrations
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A custom microarray with exon level resolution was developed to identify gross deletions or duplications. The microarray was developed using the online application eArray software (Agilent Technologies, https://earray.chem.agilent.com/earray/ ). The microarray contains approximately 60,000 interrogating oligonucleotide probes that were annotated against the human genome assembly build 37 (February 2009; NCBI37/hg19). Probes density was increased in exons and 300 bp flanking intronic sequence, with an average of 13 probes/exon. In addition, probes were placed every 2.5 Kb of intronic sequence and heavily tiled in promoter regions. Following validation runs only those probes with optimal performance were selected for the final array design.
5
Custom Target Enrichment Arrays for Hi-C
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The SureSelectXT Custom Target Enrichment Arrays were designed using the eARRAY software (Agilent Technologies). For the VCHi-C, biotinylated 120-mer RNA baits were designed to both ends of HindIII restriction fragments that contained at least 1 CCV [5 ]. A total of 1448 HindIII fragments were captured, covering 6044/7394 CCVs. For the PCHi-C, biotinylated 120-mer RNA baits were designed to both ends of HindIII restriction fragments that overlapped annotated promoters within 1 Mb of CCVs [5 ]. A total of 4049 HindIII fragments were captured, overlapping 2298 Ensembl-annotated promoters (GRCh38) [16 (link)]. A bait sequence was accepted if its GC content was between 25 and 65%, the sequence contained no more than 2 consecutive nucleotides of the same identity, and was within 330 bp of the HindIII restriction fragment end. Repetitive elements were masked using SureDesign masking tools with the highest level of stringency.
6
Transcriptomics of Adenine Metabolism in A. adeninivorans
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Based on 6,025 annotated chromosomal sequences and 36 putative mitochondrial genes oligos were designed using Agilent Technologies eArray software (design number 035454). Depending on the sequence length of the genes up to ten 60-mers per gene were created resulting in a total of 56,312 A. adeninivorans specific oligos. The microarray was produced by Agilent Technologies (Böblingen, Germany) in 8×60k format.
Overnight cultures of A. adeninivorans LS3 in YMM with nitrate were shifted to YMM containing 4 mM adenine as the sole nitrogen source and YMM with nitrate as a control, respectively. After 2 h of shaking at 30°C and 180 rpm cells were harvested and total RNA was isolated. Probe labeling and microarray hybridization (duplicates) were executed according to the manufacturer’s instructions (Agilent Technologies “One-Color Microarray-Based Gene Expression Analysis”; v6.5; Böblingen, Germany).
Analysis of microarray data was performed with the R package limma [72 ]. Raw expression values were background corrected (method “normexp”) and normalized between arrays (method “quantile”). Differentially expressed genes were detected by fitting a linear model to log2-transformed data by an empirical Bayes method [73 ]. The Bonferroni method was used to correct for multiple testing.
Overnight cultures of A. adeninivorans LS3 in YMM with nitrate were shifted to YMM containing 4 mM adenine as the sole nitrogen source and YMM with nitrate as a control, respectively. After 2 h of shaking at 30°C and 180 rpm cells were harvested and total RNA was isolated. Probe labeling and microarray hybridization (duplicates) were executed according to the manufacturer’s instructions (Agilent Technologies “One-Color Microarray-Based Gene Expression Analysis”; v6.5; Böblingen, Germany).
Analysis of microarray data was performed with the R package limma [72 ]. Raw expression values were background corrected (method “normexp”) and normalized between arrays (method “quantile”). Differentially expressed genes were detected by fitting a linear model to log2-transformed data by an empirical Bayes method [73 ]. The Bonferroni method was used to correct for multiple testing.
7
DMD Gene Enrichment Probe Library
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A customized probe library was generated using the eArray software (Agilent Technologies), as described in the user's guide. The synthetic 120-mer biotinylated oligonucleotide probes (baits) in solution were tiled along targeted intronic and exonic regions of the DMD and 3 different human control genes (FXR1, CKLF and ACTB). The genomic sequences corresponding to the 4 target genes were based on UCSC hg19-GRCh37 (chrX:31,137,336–33,357,726; chr3:180,630,234–180,695,106; chr16:66,586,466–66,600,190; chr7:55,70,372–55,66,779), respectively. To ensure capturing of intron containing pre-mRNA transcripts and low abundant transcripts, each sequence of the gene (except repeat areas) was covered generally by at least 4 baits, and the DMD promoter regions were covered on average by 8 baits. The maximum capacity of the synthesized library was up to 55K baits. The following parameters were chosen: sense strand, 1x capture-probe tiling frequency, layout strategy-centered, 20 bp overlap region between baits and avoid repeated masked regions.
8
Capture Hi-C Library Preparation and Target Enrichment
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Capture Hi-C libraries were prepared according to the protocol described in (Jäger et al., 2015 (link)). The protocol consists of two parts: Hi-C library preparation and target enrichment (Figure 1A ). A SureSelect Custom Target Enrichment Library covering a 3 Mb region in the 8p23.1 (hg19 coordinates: chr8:8,190,000–11,838,000) was designed using eArray software (Agilent). Hi-C library preparation, comprising chromatin fixation, HindIII digestion, biotin labelling, ligation, and crosslink reversal was performed as described in (Rao et al., 2014 (link)) with minor modifications described in (Jäger et al., 2015 (link)). Target enrichment was performed according to the SureSelect protocol (Agilent) with minor modifications described in (Jäger et al., 2015 (link)). We have prepared 10 capture Hi-C libraries from 5 independent batches of NA07000 cells and 4 capture Hi-C libraries from 2 independent batches of NA07056 cells. The libraries were sequenced on Illumina HiSeq 2,500 system, producing 461 million and 206 million paired-end 2 × 125 bp reads for NA07000 and NA07056, respectively.
9
Custom eel gene expression microarray
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The eel-specific gene expression microarray (4x44K) slides were custom designed with the eArray software (Agilent technologies), following MIAME guidelines [27 (link)]. A custom selection of transcripts from the de novo annotation, enhancing immune-related transcripts, was used for probe design. The arrays contained in total 41,383 probes of 60-oligonucleotide length. These probes were distributed as the following, 11,096 annotated singlets were chosen and two probes per target were added (22,192), a total of 6,397 annotated contigs with 3 probes per target (19,191) and the rest were filled with internal control probes. Settings used were based on the following: base composition methodology, best probe methodology, and designed with a 3’ bias.
10
Candida albicans mRNA Profiling by DNA Chip
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In a single pilot experiment, we tested by DNA chip hybridization if our experimental setup was suitable for the detection of significant changes in mRNA levels. For DNS chip studies, an Agilent 60-mer oligonucleotide high density array (GE 4x44K AMADID 066939 SurePrint microarray slide; Chromoscience Ltd., Gencsapati, Hungary) was constructed. Oligos were designed with the eArray software of Agilent (design number: CA5314L) based on the C_albicans_SC5314/ Assembly21 coding sequence dataset (www.candidagenome.org ). Total RNA was isolated as described above from representative tBOOH treated and untreated cultures of the WT and KO strains. Cyanine-3 (Cy3) labeled cRNA was prepared according to Agilent’s One-Color Labeling protocol (version 6.7). Prenormalised microarray data were obtained with Agilent’s Feature Extraction software (version 12.0.3.1) using default one-color high density protocol. Prenormalised data were background corrected using the normexp+offset method [47 (link)] followed by quantile normalization between arrays [48 (link)] as in Smyth [49 ].
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