Sequencing adaptor

Manufactured by Illumina

Sourced in United States

Illumina sequencing adaptors are short oligonucleotide sequences that are attached to DNA fragments during library preparation for Illumina sequencing platforms. Their core function is to provide binding sites for sequencing primers, enabling the sequencing of the DNA fragments on Illumina instruments.

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Lab products found in correlation

Nextseq 500, by Illumina (7 mentions) Hiseq 2500, by Illumina (6 mentions) Hiseq 2000, by Illumina (5 mentions) Truseq stranded mrna sample preparation kit, by Illumina (4 mentions) Miseq instrument, by Illumina (4 mentions) 2100 bioanalyzer, by Agilent Technologies (4 mentions) Qubit 2.0 fluorometer, by Thermo Fisher Scientific (4 mentions) Nanodrop, by Thermo Fisher Scientific (3 mentions) Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions) Miseq platform, by Illumina (3 mentions) Ampure xp, by Illumina (3 mentions) Ampure xp, by Beckman Coulter (3 mentions) Bioanalyzer smear analysis tool, by Agilent Technologies (3 mentions) Bioanalyzer 4200tape station, by Agilent Technologies (3 mentions) Hiseq 2000 platform, by Illumina (2 mentions) Lipofectamine rnaimax, by Thermo Fisher Scientific (2 mentions) Maxwell 16 ffpe plus lev dna purification kit, by Promega (2 mentions) Ampure xp bead, by Beckman Coulter (2 mentions) Agilent 2100 bioanalyzer, by Agilent Technologies (2 mentions) P7 barcodes, by Illumina (2 mentions)

83 protocols using sequencing adaptor

Targeted BRCA1/2 Mutation Sequencing

Cited 3 times

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Blood samples were collected and genomic DNA was extracted using the Blood Genomic DNA Extraction Kit (Shanghai Lifefeng Biotech Co., Ltd., Shanghai, China). Shrimp alkaline phosphatase enzyme was used to eliminate excess primers and deoxynucleotide triphosphates in amplification reactions. To repair the ends of the digestion reaction, bases were added. Sequencing adaptors (Illumina, San Diego, CA, USA) were ligated to the end-repaired fragments with T4 DNA ligase (Agilent, Santa Clara, CA, USA), and the fragments were then purified with magnetic beads and 80% ethanol. The HiSeq X Ten high-throughput sequencing platform (Illumina) was adopted for library sequencing, with an average sequencing depth of over 1000×. The sequencing results were aligned to the reference sequences of BRCA1 (NM_0073000) and BRCA2 (NM_000059) for mutation detection using Burrows–Wheeler Aligner.^{17 (link)} Meanwhile, the Genome Analysis Tool Kit^{18 (link)} was used to recalibrate mutation sites with ANNOVAR.^{19 (link)} The interpretation of all variants referred to the American College of Medical Genetics genetic interpretation principles.^{20 (link)}

Bao S., Sun N., Li Y., Shu J., Xu J., Zhang Y, & Qiu X. (2024). BRCA mutation status and pathological characterization of breast cancer in Zhoushan Islands, China. The Journal of International Medical Research, 52(1), 03000605231223426.

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Transcriptomic Analysis of PAX8 Knockdown

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Reverse transfection with RNAiMAX Lipofectamine (Invitrogen) using 3 different predesigned siRNA oligonulceotides (catalogs s15403, s15404, and s15405; Ambion, Invitrogen), a pool of all 3 siRNA oligonucleotides targeting PAX8, a control siRNA oligonucleotide targeting no known mammalian genes, or a no-siRNA control was performed for each cell line, as previously described (18 (link)). Samples were plated in duplicated 6-well plates, with one plate assigned for RNA-Seq and the other for Western blot confirmation. All cells were incubated for 72 hours before cells were harvested for protein or RNA. For extraction, cells were lysed using RLT buffer. Then, the lysate was homogenized using Qiashredder spin columns (Qiagen). RNA was isolated using RNeasy mini columns (Qiagen) and quantitated using a Nanodrop (Thermo Fisher Scientific), followed by Turbo DNAse treatment (Thermo Fisher Scientific) at 37°C for 30 minutes and requantification. After repairing the RNA ends, sequencing adaptors (Illumina) were ligated to the purified RNA, and the RNA was amplified for sequencing. Sequencing was performed on a HiSeq 2000 platform (Illumina). Primary sequencing data have been deposited in the GEO database, accession number GSE83101.

Elias K.M., Emori M.M., Westerling T., Long H., Budina-Kolomets A., Li F., MacDuffie E., Davis M.R., Holman A., Lawney B., Freedman M.L., Quackenbush J., Brown M, & Drapkin R. (2016). Epigenetic remodeling regulates transcriptional changes between ovarian cancer and benign precursors. JCI Insight, 1(13), e87988.

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Virus Integration Site Analysis

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VISA was performed by the Center for Cancer Research Genomics Technology Laboratory (20 (link),21 ). Briefly, genomic DNA was sheared to an average size of 400bp and subjected to linker-mediated, nested PCR using a combination of long terminal repeat (lentivirus) and linker-specific primers. Illumina sequencing adaptors were added at the same time. The library was sequenced on Illumina MiSeq using 2×150bp PE reads. Integration site junctions were mapped to hg19 human reference genome. Insertion sites are expressed as a percentage of the total reads. To compare samples within an experiment, integration sites detected in at least 2 of the samples were subjected to unsupervised hierarchical cluster analysis using R to visualize relative changes in clonal diversity.

McAbee J.H., Rath B.H., Valdez K., Young D.L., Wu X., Shankavaram U.T., Camphausen K, & Tofilon P.J. (2019). Radiation drives the evolution of orthotopic xenografts initiated from glioblastoma stem-like cells. Cancer research, 79(23), 6032-6043.

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Targeted Sequencing of Cancer Genes

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Genomic DNA was extracted from 40 μg of FFPE tumor tissue using a Maxwell 16 FFPE Plus LEV DNA Purification kit (Promega, Madison, WI, USA). Samples of 50–200 ng of extracted DNA were sheared to ~100–400 bp by sonication and were then subjected to end-repair, dA-addition, and ligation of indexed Illumina (San Diego, CA, USA) sequencing adaptors [11 (link)]. Sequencing libraries were hybridization-captured using a pool of >24 000 individually synthesized 5′-biotinylated DNA oligonucleotides (Integrated DNA Technologies, Coralville, IA, USA). These baits were designed to target ~1.5 MB of the human genome, including 4557 exons of 287 cancer-related genes, 47 introns of 19 genes frequently rearranged in cancer, and 3549 polymorphisms located throughout the genome. DNA sequencing was performed using the HiSeq instrument (Illumina) with 49 × 49 paired-end reads, targeting >500× unique median sequence coverage. Sequence data analysis and quality control measures are described in the Supplementary material, along with gene expression data analysis, support vector machine training with sequence and protein structural features, and ATM protein modeling.

Plimack E.R., Dunbrack R.L., Brennan T.A., Andrake M.D., Zhou Y., Serebriiskii I.G., Slifker M., Alpaugh K., Dulaimi E., Palma N., Hoffman-Censits J., Bilusic M., Wong Y.N., Kutikov A., Viterbo R., Greenberg R.E., Chen D.Y., Lallas C.D., Trabulsi E.J., Yelensky R., McConkey D.J., Miller V.A., Golemis E.A, & Ross E.A. (2015). Defects in DNA Repair Genes Predict Response to Neoadjuvant Cisplatin-based Chemotherapy in Muscle-invasive Bladder Cancer. European urology, 68(6), 959-967.

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Transcriptome Profiling of Notopterygium Species

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We isolated the total RNA of two Notopterygium species using the RNeasy Kit (Qiagen, Hilden, Germany) [13 (link)]. A mixed sample with equal volumes (RNAs of leaves, stems and flowers) was prepared as a single pooled RNA sample for each species. cDNA preparation and RNA-Seq were carried out using these pooled samples. Double-stranded cDNA was synthesized and sequencing adaptors were ligated according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). The ligated products were then purified with AMPureXP beads and amplified for the construction of cDNA libraries [62 (link)]. The libraries were sequenced using the Illumina HiSeq^TM 2000 (Novogene, Tianjin, China) after completion of cDNA libraries. The raw transcriptome datasets of N. incisum and N. franchetii were deposited in the NCBI Sequence Read Archive (accession numbers SRR5573673 and SRR5659683, respectively).

Jia Y., Liu M.L., Yue M., Zhao Z., Zhao G.F, & Li Z.H. (2017). Comparative Transcriptome Analysis Reveals Adaptive Evolution of Notopterygium incisum and Notopterygium franchetii, Two High-Alpine Herbal Species Endemic to China. Molecules : A Journal of Synthetic Chemistry and Natural Product Chemistry, 22(7), 1158.

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RNA-seq Library Construction and Sequencing

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Individual isolated RNA was used to construct cDNA libraries for transcriptome sequencing. In brief, mRNA was enriched using the NEBNext Poly(A) mRNA Magnetic Isolation Module (E7490, NEB, Ipswich, UK) from 3 µg of total RNA. Double-stranded cDNA was synthesized, and sequencing adaptors were ligated according to the manufacturer’s instructions (Illumina, San Diego, CA, USA). The ligated products were then purified with AMPureXP beads (Beckman Coulter, Brea, CA, USA) and were amplified for the construction of cDNA libraries. Library insert sizes ranged from 100 to 200 bp. The completed libraries were sequenced on the Illumina HiSeq 2000 platform. The original RNA-seq data was deposited into the Sequence Read Archive (SRA) of the National Centre of Biotechnology Information (NCBI) under the accession numbers SRR6015000, SRR6015001, and SRR5832159 to SRR58321162.

Duan D., Jia Y., Yang J, & Li Z.H. (2017). Comparative Transcriptome Analysis of Male and Female Conelets and Development of Microsatellite Markers in Pinus bungeana, an Endemic Conifer in China. Genes, 8(12), 393.

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Stranded RNA-Seq for Mammalian Samples

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Total RNA was used to prepare cDNA libraries with the SMARTer® Stranded Total RNA-Seq Kit v2 - Pico Input Mammalian (TaKaRa Bio USA Inc., Mountain View, CA). Briefly, 8 ng of total RNA was subjected to fragmentation followed by reverse-transcription. Illumina sequencing adaptors were attached during PCR amplification and the double-stranded cDNA was purified using AMPure XP magnetic beads. Finally, the cDNA was subjected to rRNA depletion and the stranded libraries were pre-amplified with PCR. The library size distribution was validated and quality inspected on an Agilent 2100 Bioanalyzer using a High Sensitivity DNA chip (Agilent Technologies). The quantity of each cDNA library was measured using a Qubit® 3.0 Fluorometer (Thermo Fisher Scientific). Libraries were sequenced to a read depth of >35M reads per sample using 1 × 75 bp single-end (SE) sequencing (Illumina®, San Diego, CA) on the Illumina NextSeq 500.

Michalson K.T., Macintyre A.N., Sempowski G.D., Bourland J.D., Howard T.D., Hawkins G.A., Dugan G.O., Cline J.M, & Register T.C. (2019). Monocyte Polarization is Altered by Total-Body Irradiation in Male Rhesus Macaques: Implications for Delayed Effects of Acute Radiation Exposure. Radiation research, 192(2), 121-134.

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Two-step PCR Library Preparation

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Libraries were made with a two-step PCR protocol, in which the target genomic site of interest was first amplified (Step1) with primer containing partial Ilumina sequencing adaptors followed by a second PCR with primers that contained indices and necessary Illumina sequencing adapters (Step2). Briefly, target regions were amplified with locus-specific primers (Table S2), containing universal 5′ tails on the forward (5′-CACTCTTTCCCTACACGACGCTCTTCCGATCT-3′) and reverse (5′-GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT-3′) primers. PCR amplifications were performed with EconoTaq PLUS GREEN 2X Master Mix or AccuPrime Taq DNA Polymerase, High Fidelity, according to the manufacturer protocol. Indexing of the Step1 PCR product was performed by using 0.1X volume from Step1 with indexing primers and melting at 94 °C for 2 min, followed by five cycles of 94 °C for 30 sec, 54 °C for 30 sec, and 72 °C for 40 seconds. We generated 2 × 250 reads with the Illumina MiSeq platform at the Center for Genome Sciences and Systems Biology (Washington University) or the Hartwell Center (St. Jude).

Sentmanat M.F., Peters S.T., Florian C.P., Connelly J.P, & Pruett-Miller S.M. (2018). A Survey of Validation Strategies for CRISPR-Cas9 Editing. Scientific Reports, 8, 888.

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High-throughput Chromatin Proximity Capture

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Example 6

Chromatin aggregates are cross-linked with formaldehyde and digested with the MboI restriction enzyme. The recessed 3′ ends are filled in with DNA polymerase (FIG. 14A.1). Using Klenow (3′-5′ exo), the 3′ ends are adenylated (FIG. 14A.2). Adaptors are ligated via TA-mediated ligation using DNA ligase. The adapters have 3 sections: 1) a single-stranded 5′ overhang suitable for a chromatin capture platform; 2) a double-stranded region that functions to form an adaptor and further comprises a barcode region; and 3) a 3′ T overhang for TA ligation (FIG. 14A.3). The free 5′ ends are phoshorylated and ligated to the downstream chromatin capture platform, which has multiple resolved loci each comprising binding probes that share a locus-specific barcode. Each of the adaptors are extended using the binding probes as templates to generate extension products that comprise the locus-specific barcodes. Illumina sequencing adaptors are ligated to the extension products, which are subsequently amplified and characterized by high-throughput sequencing. Based on the sequencing information, the extension products that share the same locus-specific are binned together to form a read-set. The read-sets are used to determine the order and orientation of known contigs, and thereby assemble a genome.

US10526641B2. Tagging nucleic acids for sequence assembly (2020-01-07). Dovetail Genomics, LLC [US]. Inventors: Andrew Fields [US], Paul Hartley [US], Nicholas Putnam [US], Brandon Rice [US], Jonathan Stites [US].

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Enhancer Tiling and Sequencing Workflow

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Cloned sublibraries were amplified via PCR with the primers ‘Enh-BC_Tiling-A_Stag = X_For’ (for sublibrary A and SOX10-KD library), ‘Enh-BC_Tiling-B_Stag = X_For’ (for sublibrary B), and ‘Enh-BC_Tiling_Rev.’ Illumina sequencing adaptors were added during a second round of PCR with the primers ‘i5_Indexing_For’ and ‘i7_Indexing_Rev.’ After sequencing in NovaSeq600 for 251 cycles in read 1 and 51 cycles in read 2, whole-length enhancers and random BCs were extracted from read 1 and read 2, respectively, with Cutadapt before being filtered for quality (Q > 30). Enhancer reads were mapped and linked to random BCs as previously described for CHEQ-seq 5′/intron for H3K27ac ChIP-seq regions. Following assignment, 7356 (99.2%), 7344 (99.8%), and 6773 (99.7%) sequences could be identified in the sublibraries A and B and the SOX10-KD library, respectively, with an average of 3096, 3021, and 8056 BCs per enhancer.

Mauduit D., Taskiran I.I., Minnoye L., de Waegeneer M., Christiaens V., Hulselmans G., Demeulemeester J., Wouters J, & Aerts S. (2021). Analysis of long and short enhancers in melanoma cell states. eLife, 10, e71735.

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