Hiseq2500 sequencing machine

Manufactured by Illumina

Sourced in United States

The HiSeq2500 is a high-throughput DNA sequencing machine manufactured by Illumina. It is capable of generating large amounts of sequence data through parallel sequencing of DNA fragments. The core function of the HiSeq2500 is to perform Next-Generation Sequencing (NGS) to determine the nucleotide sequence of DNA samples.

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Lab products found in correlation

Truseq stranded mrna library prep kit, by Illumina (3 mentions) Rneasy plant mini kit, by Qiagen (2 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (2 mentions) Dragen bio it platform version 3, by Illumina (1 mentions) Sure select human all exon kit v6, by Illumina (1 mentions) Dneasy blood tissue kit, by Qiagen (1 mentions) Sureselect human all exon v6 kit, by Agilent Technologies (1 mentions) Twist human core exome kit, by Twist Bioscience (1 mentions) Xgen exome research panel v2 kit, by Integrated DNA Technologies (1 mentions) Nanodrop nd 1000 spectrophotometer, by Thermo Fisher Scientific (1 mentions) M mlv reverse transcriptase, by Promega (1 mentions) Tri reagent solution, by Thermo Fisher Scientific (1 mentions) Agilent 2100 rna nanochip, by Agilent Technologies (1 mentions) Dna free kit, by Thermo Fisher Scientific (1 mentions) Nextera xt dna library preparation kit, by Illumina (1 mentions) 2100 bioanalyzer, by Agilent Technologies (1 mentions) Nebnext dsdna fragmentase, by New England Biolabs (1 mentions) Kapa hyper prep kit, by Illumina (1 mentions) Rnaeasy plant mini kit, by Qiagen (1 mentions)

Spelling variants of this product

Illumina sequencing machine (5 citations)

10 protocols using hiseq2500 sequencing machine

X-Chromosome Inactivation Profiling by WES

Cited 1 time

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DNA samples of patients demonstrating skewed X-chromosome inactivation were examined by WES, followed by X chromosome-focused bioinformatics analysis (Genomics Unit, Center for Cancer Research, Sheba Medical Center).
Exome sequencing was performed using a Sure Select Human All Exon kit V6 on a HiSeq2500 sequencing machine (Illumina, San Diego, CA, USA). For each sample, paired end reads (2 × 150 bp) were obtained and processed. The Illumina Dragen Bio-IT Platform version 3.8 was used to align reads to the human reference genome (hg38) based on the Smith–Waterman algorithm [13 (link)], as well as to call variants based on the GATK variant caller version 3.7 [14 (link)]. Additional variants were called with Freebayes version 1.2.0 [15 ]. Variant annotation was performed using KGG-Seq version 1.2 [16 (link)]. Further annotation and filtration steps were performed by in-house scripts using various additional datasets.

Dardik R., Avishai E., Lalezari S., Barg A.A., Levy-Mendelovich S., Budnik I., Barel O., Khavkin Y., Kenet G, & Livnat T. (2021). Molecular Mechanisms of Skewed X-Chromosome Inactivation in Female Hemophilia Patients—Lessons from Wide Genome Analyses. International Journal of Molecular Sciences, 22(16), 9074.

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Total RNA Extraction and RNA-seq Library Prep

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Total RNA was extracted using the RNeasy Plant Mini Kit (QIAGEN) with dsDNase treated following the manufacturer’s instruction. The RNA concentration was measured using Qubit® 2.0 Fluorometer (Invitrogen). 4 μg total RNA was used to prepare RNA-seq libraries using the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, US). Single-end sequencing was performed on Illumina HiSeq 2500 sequencing machine at the Next-Generation Sequencing Core of the Salk Institute for Biological Studies.

Huang J., Zhao X, & Chory J. (2019). The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress. Cell reports, 29(12), 4186-4199.e3.

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Genomic DNA Extraction and RAD Sequencing

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For DNA extraction, we used the QIAGEN DNeasy Blood Tissue Kit (QIAGEN, Valencia, CA, USA), following the manufacturer's protocol. RNase A was added to get RNA‐free genomic DNA. Original RAD libraries (Andrews et al., 2016) were prepared as in Pedersen et al. (2018). We used the restriction enzyme SbfI on 250 ng of DNA per sample. After adapter ligation, we pooled samples with similar quality, based on agarose gel analyses, into separate sublibraries that were sheared separately through sonication. More fragmented libraries were sheared for a shorter amount of time than less fragmented libraries to retain larger RAD fragments in those individuals. We single‐end sequenced the libraries on the Illumina HiSeq 2,500 sequencing machine at the Institute of Molecular Biology, University of Oregon. We demultiplexed the files and performed crude quality filtering using process_radtags in STACKS v. 1.34 (Catchen et al., 2013) with default settings. We also checked the fastq data using fastqc (Andrews, 2010) and used AdapterRemoval v. 2 (Schubert et al., 2016) to remove any possible adapter contamination in the reads. We subsequently removed all reads from which possible adapter sequence had been cut. After these steps, we mapped the resulting fastQ files to the cow reference genome bosTau8 using bwa mem with default settings (Li & Durbin, 2009).

Garcia‐Erill G., Kjær M.M., Albrechtsen A., Siegismund H.R, & Heller R. (2020). Vicariance followed by secondary gene flow in a young gazelle species complex. Molecular Ecology, 30(2), 528-544.

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Exome Sequencing Bioinformatics Pipeline

Cited 2 times

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Since the sequencing itself was not performed at SMC, DNA samples were sent to one of three approved service laboratories. One of the following three capture kits were used for ES: Twist Human Core Exome Kit (Twist Bioscience, San Francisco, CA, USA); SureSelect Human All Exon Kit V6 (Agilent, Santa Clara, CA, USA); or xGen Exome Research Panel V2 Kit (Integrated DNA Technologies, Coralville, Iowa, USA). In all cases, sequencing runs were performed on a HiSeq2500 sequencing machine (Illumina, San Diego, CA, USA).
Bioinformatics analysis was carried out at the Genomics Unit of the Sheba Cancer Research Center. Reads were aligned to the UCSC human reference assembly (hg19) with BWA-MEM algorithm (BWA v.0.7.15)¹⁶. Variants were called by applying GATK variant caller version 3.8 using guidelines recommended by the Broad Institute^{17 (link)}, and annotated with KGGSEQ^{18 (link)}. Further annotation and filtration steps were taken using various public databases, the Human Gene Mutation Database (HGMD) and internal databases by application of in-house custom scripts. For 3 families, ES was analyzed in another laboratory (outside SMC).
Variants were interpreted according to the American College of Medical Genetics and Genomics (ACMG) guidelines^{19 (link)}, and were classified as pathogenic, likely-pathogenic, variants of uncertain significance (VUS), likely benign or benign.

Pode-Shakked B., Barel O., Singer A., Regev M., Poran H., Eliyahu A., Finezilber Y., Segev M., Berkenstadt M., Yonath H., Reznik-Wolf H., Gazit Y., Chorin O., Heimer G., Gabis L.V., Tzadok M., Nissenkorn A., Bar-Yosef O., Zohar-Dayan E., Ben-Zeev B., Mor N., Kol N., Nayshool O., Shimshoviz N., Bar-Joseph I., Marek-Yagel D., Javasky E., Einy R., Gal M., Grinshpun-Cohen J., Shohat M., Dominissini D., Raas-Rothschild A., Rechavi G., Pras E, & Greenbaum L. (2021). A single center experience with publicly funded clinical exome sequencing for neurodevelopmental disorders or multiple congenital anomalies. Scientific Reports, 11, 19099.

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Transcriptome Analysis of Mantle Tissue Dynamics

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Total RNA was extracted from the mantle tissue of 4 individuals per condition at each time point using TRI Reagent® Solution (Life Technologies) according to manufacturer’s instructions (22 libraries in total). RNA from the four individuals was then pooled for each time point separately. RNA quality and concentration were determined using an Agilent 2100 RNA Nanochip (Agilent, Santa Clara, CA, USA) and by NanoDrop ND-1000 Spectrophotometer (260/280 nm, NanoDrop Technologies, Wilmington, DE, USA), respectively. Samples were then treated with a DNA-free Kit (Ambion, Austin, TX) to remove genomic DNA, and reverse transcription was carried out using 500 ng of total RNA, random hexamers, and MMLV reverse transcriptase (Promega) according to manufacturer’s protocols. The production of Illumina libraries for mRNA-seq and the transcriptome sequencing using Illumina HiSeq™ 2500 sequencing machine (HiSeq 100 pair-ends) was conducted by the Genome Analysis Centre (Norwich, UK), as described in [24 (link)].

Artigaud S., Richard J., Thorne M.A., Lavaud R., Flye-Sainte-Marie J., Jean F., Peck L.S., Clark M.S, & Pichereau V. (2015). Deciphering the molecular adaptation of the king scallop (Pecten maximus) to heat stress using transcriptomics and proteomics. BMC Genomics, 16, 988.

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Whole Exome Sequencing of Pool Samples

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Whole Exome Sequencing (WES) was carried out at Genomic lab, Rehman Medical Institute (RMI), Hayatabad, Peshawar. Paired end libraries of pool samples were prepared using Illumina Nextera XT DNA library preparation kit. Quantified DNA libraries were sequenced using HiSeq2500 sequencing machine (Illumina, San Diego, CA, USA).

Jan A., Z.a.k.i.u.l.l.a.h., Ali S., Muhammad B., Arshad A., Shah Y., Bahadur H., Khan H., Khuda F., Akbar R, & Ijaz K. (2023). Decoding type 2 diabetes mellitus genetic risk variants in Pakistani Pashtun ethnic population using the nascent whole exome sequencing and MassARRAY genotyping: A case-control association study. PLOS ONE, 18(1), e0281070.

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Bacterial mRNA Purification and Transcriptome Sequencing

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Removal of 16S and 23S rRNAs from total RNA was performed with the MICROBExpress bacterial mRNA puriﬁcation kit (Ambion) according to manufacturer’s protocol (67 (link)). RNA concentrations and quality were determined by using the Bioanalyzer 2100 (Agilent). For whole-transcriptome sequencing (RNA-seq), cDNA libraries were prepared by the Illumina mRNA-seq TruSeq protocol according to the manufacturer’s protocol without the poly(A) isolation stage. Sequencing was performed with an Illumina HiSeq 2500 sequencing machine. Sequencing generated 20 to 28 million raw reads per sample, of which 2 to 6 million were mapped to protein-encoding genes. The RNA-seq data were analyzed by a standard protocol based on the number of reads per kilobase of coding sequence length per million reads analyzed (RPKM) (68 (link)).

Huja S., Oren Y., Biran D., Meyer S., Dobrindt U., Bernhard J., Becher D., Hecker M., Sorek R, & Ron E.Z. (2014). Fur Is the Master Regulator of the Extraintestinal Pathogenic Escherichia coli Response to Serum. mBio, 5(4), e01460-14.

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Total RNA Extraction and RNA-seq Library Prep

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Huang J., Zhao X, & Chory J. (2019). The Arabidopsis Transcriptome Responds Specifically and Dynamically to High Light Stress. Cell reports, 29(12), 4186-4199.e3.

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High-throughput DNA library sequencing

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Samples were fragmented using NEBNext dsDNA Fragmentase (New England BioLabs, UK) and the libraries were prepared using the KAPA Hyper Prep Kit Illumina (KAPA Biosystems, USA) following the manufacturer's instructions with only one modification: 96 different eight-base barcoded adapters were used instead of the Illumina original ones, in order to pool more libraries with different barcodes into one sequencing lane. Two sequencing lanes were used for sequencing, so the 96 libraries were pooled into two groups, each of them comprised of 48 libraries. The libraries were sequenced using a HiSeq2500 sequencing machine (Illumina, USA) in rapid run mode with 100 bp paired-end reads by the Genomics Sequencing Laboratory (GSL) at the University of California, Berkeley, USA.

Fanelli V., Ngo K.J., Thompson V.L., Silva B.R., Tsai H., Sabetta W., Montemurro C., Comai L, & Harmer S.L. (2021). A TILLING by sequencing approach to identify induced mutations in sunflower genes. Scientific Reports, 11, 9885.

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Arabidopsis RNA-seq Transcriptomic Analysis

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The RNA-seq data utilized in this research was obtained from a previous work published in Cell Reports [57 (link)]. Briefly, 7-day-old Arabidopsis seedlings (Columbia, Col-0) were grown in MS media and subjected to continuous high light (HL, 1200 mmol m⁻² s⁻¹), with control plants kept under the growth light condition (GL, 60 mmol m⁻² s⁻¹). Samples were collected at time periods of 0.5 h, 6 h, 12 h, 24 h, 48 h, and 72 h, after which HL plants were returned to the GL condition and a recovery sample was collected after 14 h. Total RNA was extracted using the RNAeasy Plant Mini Kit (QIAGEN), and RNA-seq libraries were prepared using 4 mg of total RNA and the TruSeq Stranded mRNA Library Prep Kit (Illumina, San Diego, CA, USA). Subsequently, single-end RNA-seq was generated on an Illumina HiSeq 2500 sequencing machine at the Next-Generation Sequencing Core of the Salk Institute for Biological Studies. The raw counts of each gene were determined using the BRB Digital Gene Expression (BRB-DGE) program (https://arraytools.github.io/bdge/).

, & Albaqami M. (2023). The Splicing Factor SR45 Negatively Regulates Anthocyanin Accumulation under High-Light Stress in Arabidopsis thaliana. Life, 13(6), 1386.

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