Hiseq 2500

Manufactured by Illumina

Sourced in United States, China, Germany, United Kingdom, Canada, Switzerland, Sweden, Japan, Australia, France, India, Hong Kong, Spain, Cameroon, Austria, Denmark, Italy, Singapore, Brazil, Finland, Norway, Netherlands, Belgium, Israel

The HiSeq 2500 is a high-throughput DNA sequencing system designed for a wide range of applications, including whole-genome sequencing, targeted sequencing, and transcriptome analysis. The system utilizes Illumina's proprietary sequencing-by-synthesis technology to generate high-quality sequencing data with speed and accuracy.

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Lab products found in correlation

Agilent 2100 bioanalyzer, by Agilent Technologies (602 mentions) Trizol reagent, by Thermo Fisher Scientific (469 mentions) 2100 bioanalyzer, by Agilent Technologies (401 mentions) Rneasy mini kit, by Qiagen (380 mentions) Trizol, by Thermo Fisher Scientific (365 mentions) Hiseq 4000, by Illumina (240 mentions) Hiseq 2000, by Illumina (240 mentions) Nextseq 500, by Illumina (202 mentions) Bioanalyzer, by Agilent Technologies (194 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (180 mentions) Novaseq 6000, by Illumina (166 mentions) Nanodrop 2000, by Thermo Fisher Scientific (155 mentions) Ampure xp bead, by Beckman Coulter (130 mentions) Nanodrop, by Thermo Fisher Scientific (127 mentions) Qubit fluorometer, by Thermo Fisher Scientific (117 mentions) Truseq rna sample preparation kit, by Illumina (116 mentions) Truseq small rna sample prep kit, by Illumina (113 mentions) Rneasy kit, by Qiagen (112 mentions) Truseq rna sample preparation kit v2, by Illumina (105 mentions) Nebnext ultra rna library prep kit for, by Illumina (103 mentions)

11 613 protocols using hiseq 2500

Benchmark Callsets for GIAB Samples

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We used sequencing reads and benchmark callsets of the seven individuals currently in GIAB. HG001 (a female of Utah/European ancestry) and HG002 (Ashkenazi Jewish son) samples were sequenced by Illumina HiSeq 2500 in Rapid Mode (v1) with 2x148bp read length and ~50x coverage, and were aligned to GRCh38 by BWA-MEM version 0.7.17 [46] . The Ashkenazi Jewish parent samples, HG003 and HG004, were sequenced with Illumina HiSeq 2500 in Rapid mode (v2) with 2x250 paired-end reads and ~40-50x coverage, and were aligned to GRCh38 using Novoalign version 3.02.07 (www.novocraft.com/products/novoalign). HG005, the Chinese son sample, was used only for imputation evaluation based on the benchmark callset. The Chinese parent samples HG006 and HG007 were sequenced by Illumina HiSeq 2500 in Rapid mode (v1) with 2x148 paired end reads to 100x coverage, mapped to GRCh38 with BWA-MEM, and downsampled to ~40x coverage to match the coverage of other samples using samtools v1.6 [37] .

Yun T., Li H., Chang P., Lin C., Carroll A., & McLean C.Y. (2020). Accurate, scalable cohort variant calls using DeepVariant and GLnexus.

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Graphene Oxide Exposure Effects on Nematode Transcriptome

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It was reported that prolonged exposure (from L1-larvae to young adults) to GO at concentrations more than 0.5 mg L⁻¹ could cause toxicity on the functions of both primary targeted organs and secondary targeted organs.^{57 (link)} The 1 mg L⁻¹ was selected as working concentration for GO exposure for Illumina HiSeq2500 sequencing. For each RNA sample, total RNAs were obtained from control or GO exposed wild-type nematodes using Trizol (Invitrogen, UK) according to manufacturer's protocol. Total genomic DNA was removed using DNase I (New England Biolabs), and RNA purity was assessed using Nanodrop2000. Total RNA was subject to ribosomal RNA depletion according to manufacturer's protocol of Ribo-Minus kit. cDNA libraries were generated using TruSeq RNA Sample Prep Kit v2 (Illumina). Each library was loaded into one lane of Illumina HiSeq2500 for 2 × 125 bp pair-end sequencing, followed by on-board cluster generation on a Rapid Run pair-end flow cell and subsequent 125 cycles sequencing (v3 sequencing kit) according to manufacturer's instructions (HiSeq 2500, Illumina). Three independent biological replicates were performed.

Shi L., Jia X., Guo T., Cheng L., Han X., Wu Q, & Wang D. (2019). A circular RNA circ_0000115 in response to graphene oxide in nematodes. RSC Advances, 9(24), 13722-13735.

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RNA-seq Library Preparation and Analysis

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RNA sequencing libraries were prepared with Kapa RNA mRNA HyperPrep kit (Kapa Biosystems, USA) according to the manufacturer’s protocol. The sequencing libraries were validated with the Agilent Bioanalyzer DNA High Sensitivity Kit and quantified with Qubit. The libraries were sequenced on Illumina HiSeq 2500 with SR V4 Kit with the single read mode of 51cycle of read1 and 7 cycles of index read. Real-time analysis (RTA) 2.2.38 software was used to process the image analysis and base calling.
RNA sequencing data analyses were performed using the HiSeq 2500 (Illumina, USA) according to manufacturer’s instructions. Briefly, 500 ng of RNA was converted into cDNA library, which was then sequenced using Illumina Hiseq2500 with single read 40 bp based on manufacturer’s instructions. Raw sequence reads were mapped to the human genome (hg19) using STAR_2.5.3a [71 (link)] and the frequency of genes was counted using HTSeq-0.6.1p1 [72 (link)]. The raw counts were then normalized using the trimmed mean of M values (TMM) method and compared using Bioconductor package “edgeR 3.16.5” [73 (link)]. Reads per kilobase per million (RPKM) mapped were also calculated from the raw counts. Differentially expressed genes were identified if RPKM ≥ 1 in at least one sample, fold change ≥ 2, and P ≤ 0.05.

Pangeni R.P., Yang L., Zhang K., Wang J., Li W., Guo C., Yun X., Sun T., Wang J, & Raz D.J. (2020). G9a regulates tumorigenicity and stemness through genome-wide DNA methylation reprogramming in non-small cell lung cancer. Clinical Epigenetics, 12, 88.

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De novo Genome Sequencing and Assembly

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De novo reference genome sequence was produced by interleaving Illumina MiSeq and HiSeq2500 sequence data (Illumina Inc., San Diego, CA, United States). DNA sequencing and assembly was performed as a service by the Earlham Institute. De novo genome sequencing and assembly was performed using PCR free paired-end (PE) and long mate pair (LMP) sequencing. After DNA sample QC, line C was selected for sequencing and reference genome assembly. One PCR free PE library was constructed from gDNA, and sequenced on one lane of an Illumina HiSeq2500 in rapid run-mode (v2) using 250 bp PE reads. LMP sequencing was also conducted using one set of Nextera libraries (Illumina) from gDNA, and sequenced on one lane of an Illumina MiSeq with 250 bp PE reads. After data QC and assembly of the high coverage PE library, LMP libraries were mapped to determine their suitability for assembly improvement. Three additional libraries were selected and re-sequenced to a higher depth of coverage on a single lane of an Illumina HiSeq2500 in rapid run-mode, to again yield 250 bp PE reads.

Bell L., Chadwick M., Puranik M., Tudor R., Methven L., Kennedy S, & Wagstaff C. (2020). The Eruca sativa Genome and Transcriptome: A Targeted Analysis of Sulfur Metabolism and Glucosinolate Biosynthesis Pre and Postharvest. Frontiers in Plant Science, 11, 525102.

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Bulk and Single-cell RNA-Seq Protocols

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For bulk sequencing, we added fatbody-free gonads to RNeasy 96 kit 350 μl RLT buffer mixed two times for 30 s, froze in a dry ice-ethanol bath and stored at −80 °C. We extracted total RNA from gonad samples using RNeasy 96 kit following the manufacturer’s protocol. We quantified RNA using the Quant-iT RiboGreen quantification kit and used 200 ng of total RNA for library preparation^{84 (link)}. We spiked in External RNA Control Consortium (ERCC) spike-ins pools 78 A and 78B (transcribed from a certified standard reference) prior to fragmentation. For multiplexing, we used eight different TruSeq v2 kit barcoded adaptors. We sequenced stranded multiplexed libraries using a single-ended 50 bp strategy and generated RNA-Seq profiles of biological quadruplicates on a HiSeq Illumina 2500.
For single-cell sequencing, we loaded dissociated cells (6 K for Replicates 1 and 2, 12 K for replicate 3) onto the 10X Chromium system for barcoding and library preparation following the user guide for Single Cell 3′ Reagent Kits v2. We quantified libraries with Quant-iT PicoGreen and confirmed 300–500 bp insert sizes on a TapeStation 2200. We generated scRNA-Seq profiles in biological triplicate pools on the 10X Chromium System and sequenced (Read1 = 26 bp, Read2 = 98 bp and Readi7 = 8 bp) on a HiSeq Illumina 2500.

Mahadevaraju S., Fear J.M., Akeju M., Galletta B.J., Pinheiro M.M., Avelino C.C., Cabral-de-Mello D.C., Conlon K., Dell’Orso S., Demere Z., Mansuria K., Mendonça C.A., Palacios-Gimenez O.M., Ross E., Savery M., Yu K., Smith H.E., Sartorelli V., Yang H., Rusan N.M., Vibranovski M.D., Matunis E, & Oliver B. (2021). Dynamic sex chromosome expression in Drosophila male germ cells. Nature Communications, 12, 892.

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Bacterial Whole Genome Sequencing

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For each bacterial culture, a single colony was sub-cultured and DNA was extracted from the sub-cultured plate using a mechanical lysis step on FastPrep homogeniser (MPBiomedicals, Santa Ana, CA) followed by extraction with the Quickgene-mini80 device (Autogen Inc, Holliston, MA), and sequenced at the Wellcome Trust Centre for Human Genetics, Oxford. Six hundred isolates were sequenced on the Illumina HiSeq 2500 platform (San Diego, California, USA), with paired-end reads 150 base pairs long. A further 417 isolates were sequenced for an earlier study, 26 on with Illumina HiSeq 2500, and 391 on the HiSeq 2000 platform, with paired-end reads of 99 base pairs.

Young B.C., Wu C.H., Charlesworth J., Earle S., Price J.R., Gordon N.C., Cole K., Dunn L., Liu E., Oakley S., Godwin H., Fung R., Miller R., Knox K., Votintseva A., Quan T.P., Tilley R., Scarborough M., Crook D.W., Peto T.E., Walker A.S., Llewelyn M.J, & Wilson D.J. (2021). Antimicrobial resistance determinants are associated with Staphylococcus aureus bacteraemia and adaptation to the healthcare environment: a bacterial genome-wide association study. Microbial Genomics, 7(11), 000700.

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Dual-Platform Transcriptome Profiling of Callus

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After grinding the selected callus materials of 10 time points with liquid nitrogen, total RNA was extracted by Trizol and stored at −80 °C for subsequent sequencing on PacBio Sequel and Illumina Hiseq 2500 platforms, which were entrusted to us by Novogene Co., Ltd. To obtain clean reads, high-throughput sequencing from a large amount of raw data was obtained, from which the adaptor sequence, primer sequence, and low-quality reads were removed. The LoRDEC (Salmela and Rivals, 2014 (link)) software was used to correct the PacBio Sequel data with the Illumina Hiseq 2500 data. The CD-HIT (Fu et al., 2012 (link)) software was used to de-redundant the corrected consensus sequence, and the obtained transcript set was applied as the reference sequence (ref) for subsequent analysis. Then, the clean reads of each sample obtained by Illumina sequencing were compared to the reference to determine the transcripts detected in each sample.

Liu H., Huang C., Li Q., Wang M., Xiao S., Shi J., He Y., Wen W., Li L, & Xu D. (2022). Genome-Wide Identification of Genes Related to Biosynthesis of Phenolic Acid Derivatives in Bletilla striata at Different Suspension Culture Stages. Frontiers in Plant Science, 13, 875404.

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Exploring Immune Regulation through lncRNA

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Total RNA from each sample was used to construct cDNA libraries using the VAHTSTM Total RNA‐Seq (H/M/R) for high‐throughput sequencing. In brief, the RNA was reverse transcribed into first‐strand cDNA, followed by second‐strand cDNA synthesis, end repair, dA tailing and adaptor ligation. The digested products were purified using VAHTSTM DNA Clean Beads, and PCR amplified and sequenced using Illumina HiSeq 2500 (San Diego, CA, USA). The tagged cDNA libraries were paired‐end sequenced using an Illumina HiSeq 2500 with 51 plus 7 cycles by Dianxi Biotechnology, Shanghai, China. To fully understand the biological functions of lncRNAs related to immunity and inflammation, we performed Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway enrichment analysis. Coexpression network analysis was performed using Comparative Co‐Expression Network Construction and Visualization Tool (CoExpNetViz, Chicago, IL, USA).

Wen J., Liu J., Jiang H., Wan L., Xin L., Sun Y., Zhang P., Sun Y., Zhang Y., Du X., Wang X, & Wang J. (2020). lncRNA expression profiles related to apoptosis and autophagy in peripheral blood mononuclear cells of patients with rheumatoid arthritis. FEBS Open Bio, 10(8), 1642-1654.

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Executing Comprehensive cfDNA Analysis in PDX Models

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For LuCaP PDX mouse plasma samples, NGS libraries were prepared with 50ng input cfDNA. Illumina NGS sequencing libraries were prepared with the KAPA hyperprep kit, adopting nine cycles of amplification, and purified using lab standardized SPRI beads. We used KAPA UDI dual indexed library adapters. Library concentrations were balanced and pooled for multiplexing and sequenced using the Illumina HiSeq 2500 at the Fred Hutch Genomics Shared Resources (200 cycles) and Illumina NovaSeq platform at the Broad Institute Genomics Platform Walkup-Seq Services using S4 flow cells (300 cycles). To match with Illumina HiSeq 2500 data, truncated 200 cycles FASTQ files were generated (100 bp paired end reads).
Clinical patient plasma samples collected at University of Washington (UW cohort) were submitted to the Broad Institute Blood Biopsy Services. Briefly, cfDNA was extracted from 2 mL double-spun plasma and ultra-low-pass whole genome sequencing (ULP-WGS) to approximately 0.2x coverage was performed. The ichorCNA pipeline was used to estimate tumor DNA content (i.e., tumor fraction, see below). Forty-seven samples (from 31 patients) had either ≥ 5% tumor fraction or ≥ 2% tumor fraction with AR amplification observed in ichorCNA and were subsequently sequenced to deeper WGS coverage (~20x).

De Sarkar N., Patton R.D., Doebley A.L., Hanratty B., Adil M., Kreitzman A.J., Sarthy J.F., Ko M., Brahma S., Meers M.P., Janssens D.H., Ang L.S., Coleman I.M., Bose A., Dumpit R.F., Lucas J.M., Nunez T.A., Nguyen H.M., McClure H.M., Pritchard C.C., Schweizer M.T., Morrissey C., Choudhury A.D., Baca S.C., Berchuck J.E., Freedman M.L., Ahmad K., Haffner M.C., Montgomery R.B., Corey E., Henikoff S., Nelson P.S, & Ha G. (2022). Nucleosome Patterns in Circulating Tumor DNA Reveal Transcriptional Regulation of Advanced Prostate Cancer Phenotypes. Cancer Discovery, 13(3), 632-653.

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Transcriptome Profiling by RNA-seq

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Total RNA quality was assessed by spectrophotometer (NanoDrop, Thermo Fisher Scientific Inc., Waltham, MA) and the Agilent 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). mRNA was purified from 1 μg of total RNA using poly(A) tail selection. RNA sequencing libraries were generated using the TruSeq RNA Sample Prep Kit (Illumina, San Diego, CA) with multiplexing primers, according to the manufacturer’s protocol. The cDNA library was constructed with average inserts of 275 bp, with non-stranded library preparation. Fragment size distributions were assessed using the Bioanalyzer 2100 and the DNA high-sensitivity chip. Library concentrations were measured using the KAPA Library Quantification Kit (Kapa Biosystems Inc., Wilmington, MA) and run on the Illumina HiSeq2500. Sequencing was performed via a paired-end 150 cycle rapid run on 2 lanes of the Illumina HiSeq2500, generating more than 300 million pairs of reads as desired.

Udy D.B., Voorhies M., Chan P.P., Lowe T.M, & Dumont S. (2015). Draft De Novo Transcriptome of the Rat Kangaroo Potorous tridactylus as a Tool for Cell Biology. PLoS ONE, 10(8), e0134738.

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