Adapter sequences

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Illumina adapter sequences are short, synthetic DNA fragments that are designed to be ligated to the ends of DNA fragments during library preparation for Illumina sequencing platforms. These adapters serve as a handle, allowing the DNA fragments to be captured, amplified, and sequenced. The core function of Illumina adapter sequences is to facilitate the sequencing process by providing a standardized interface between the DNA samples and the sequencing instrumentation.

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Lab products found in correlation

Nanodrop 2000, by Thermo Fisher Scientific (2 mentions) Qubit dsdna hs assay kit, by Thermo Fisher Scientific (2 mentions) Immunoseq assay, by Adaptive Biotechnologies (1 mentions) Nextera xt index kit, by Illumina (1 mentions) Miseq platform, by Illumina (1 mentions) Agencourt ampure xp bead, by Beckman Coulter (1 mentions) Nebnext library quant kit, by New England Biolabs (1 mentions) Q5 high fidelity dna polymerase, by New England Biolabs (1 mentions) Qiaamp dna micro kit, by Qiagen (1 mentions) Chromium system, by 10x Genomics (1 mentions) Dynabeads myone silane magnetic beads, by Thermo Fisher Scientific (1 mentions) Chromium controller, by 10x Genomics (1 mentions) Nextseq 500 high output flow cell, by Illumina (1 mentions) Agilent 4200 tapestation, by Agilent Technologies (1 mentions)

Spelling variants of this product

Illumina adapters (41 citations) TruSeq adapter sequences (20 citations) Overhang adapter sequences (8 citations) Nextera adapter sequences (7 citations) Adapter overhang nucleotide sequences (6 citations) Flowcell adapter sequences (6 citations) Adapter overhang sequences (3 citations) NexteraXT adapter sequences (2 citations) Standard adapter sequences (2 citations) Paired-end adapter sequences (2 citations)

6 protocols using adapter sequences

Profiling Immune Receptor Repertoire

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The sample data was generated using the immunoSEQ assay (Adaptive Biotechnologies, Seattle, WA). The somatically rearranged locus CDR3 region was amplified from genomic DNA using bias-controlled multiplex polymerase chain reaction (PCR) amplification [25 (link),26 (link)]. Specifically, the first PCR consists of forward and reverse amplification primers specific for every V and J gene segment, and it amplifies the hypervariable CDR3 of the immune receptor locus. The second PCR adds a proprietary barcode sequence (Adaptive Biotechnologies, Seattle, WA, USA) and Illumina adapter sequences (Illumina, San Diego, CA, USA) [26 (link),27 (link)]. CDR3 libraries were sequenced on an Illumina instrument according to the manufacturer’s instructions.

Kim B.J., Youn D.H., Kim Y, & Jeon J.P. (2020). Characterization of the TCR β Chain CDR3 Repertoire in Subarachnoid Hemorrhage Patients with Delayed Cerebral Ischemia. International Journal of Molecular Sciences, 21(9), 3149.

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Bacterial 16S rRNA Gene Sequencing

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Oral DNA samples (5 ng/μl) were amplified in PCR reactions targeting the V3-V5 (16 (link)) region of the bacterial 16S ribosomal RNA (rRNA) gene. The initial PCR utilized custom primers ligated with overhang Illumina adapter sequences (Integrated DNA Technologies, Inc. Coralville, IA, USA). Amplicons were purified using Agencourt AMPure XP beads (Beckman Coulter, Brea, CA, USA). To allow for multiplexing, a second PCR was run to attach unique indices to each end of 16S amplicons using the Nextera XT Index Kit (Illumina, San Diego, CA, USA). Bar-coded amplicons were purified, quantified by Nanodrop 2000 (ThermoFisher, Waltham, MA, USA), normalized to equal concentrations, pooled, and checked on agarose gels for library sizes. Bar-coded libraries (50ul of 100 ng/μl) were assayed by 2 x 300 paired-end sequencing using the 600-cycle v3 kit on the MiSeq platform (Illumina) at the University of Hawaii.

Hernandez B.Y., Zhu X., Risch H.A., Lu L., Ma X., Irwin M.L., Lim J.K., Taddei T.H., Pawlish K.S., Stroup A.M., Brown R., Wang Z., Wong L.L, & Yu H. (2021). Oral Cyanobacteria and hepatocellular carcinoma. Cancer epidemiology, biomarkers & prevention : a publication of the American Association for Cancer Research, cosponsored by the American Society of Preventive Oncology, 31(1), 221-229.

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Nextera Adapter Addition to PAM Libraries

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Add Nextera adapters to the cleaved PAM libraries and controls.
Add Illumina indices and binding domains.
With the Nextera adapters on the PAM library, Illumina indices and binding domains can be attached using PCR. To run more than one library on a single sequencing lane, a unique pair of primers must be used for each sample in order to attach a unique pair of i5 and i7 indices to each sample. A list of the Nextera indices and the oligos that they are embedded within are provided by Illumina in their technical support document “Illumina Adapter sequences” under the file heading “Illumina Nextera Adapters.” The two pairs of indexing oligos from this table that we used to elucidate the NmeCas9 PAM are provided in the appendix (RL135 (i5) and TJpr422/423 (i7)).

Maxwell C.S., Jacobsen T., Marshall R., Noireaux V, & Beisel C.L. (2018). A detailed cell-free transcription-translation-based assay to decipher CRISPR protospacer-adjacent motifs. Methods (San Diego, Calif.), 143, 48-57.

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Two-Step PCR Library Preparation

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Outputs from the microfluidic chip were subjected to DNA extraction using the QIAamp DNA Micro Kit (QIAGEN, Germany). In parallel, five hundred WBCs were counted and subjected to DNA extraction. After DNA quantification with Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific, USA), a 2-step PCR library preparation was performed with Q5 High-Fidelity DNA Polymerases (NEB, USA).
The first PCR serves to amplify target exon regions and introduce a 10-bp diversifier sequence and a portion of sequencing adapters (i.e. P5 and P7 adapters) to the amplicon library. The diversifier sequence is a sequence of ten random nucleotides, which was designed to increase the sequence diversity of libraries for accurate cluster identification by Illumina sequencer. The second PCR serves to extend the remaining portion of Illumina Adapter Sequences (Illumina, USA). Primer sequences are listed in S1 and S2 Tables. Libraries were then purified by magnetic SeqCap EZ purification beads (Roche, Switzerland) and then quantified with NEBNext Library Quant Kit (NEB, USA). Libraries were multiplexed in equimolar amounts and sequenced with MiSeq sequencing kit v2, according to Illumina’s guidelines. In general, 12 pM multiplexed library and 5% phiX spike-in were loaded.

Wong V.C., Ko J.M., Lam C.T, & Lung M.L. (2017). Succinct workflows for circulating tumor cells after enrichment: From systematic counting to mutational profiling. PLoS ONE, 12(5), e0177276.

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ClickSeq-based Reverse Transcription

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AMT-crosslinked RNA was directly used as template during reverse transcription. Standard ClickSeq protocol (35 ) was carried out. In brief, 250 ng of templated RNA was supplemented with a mixture of azido-NTP:dNTP (1:5 molecular ratio) during random primed reverse transcription. This is followed by “Click-ligation” (36 (link)) with a 5′-hexynyl-functionalized Illumina adapter (IDT). The adapter ligated cDNA fragments were subsequently PCR amplified to complete Illumina adapter sequences and to incorporate sample indexes/barcodes. The ClickSeq libraries of non-crosslinked FHV RNAs were constructed the same way as for the control.

Zhou Y, & Routh A.L. (2024). Bipartite viral RNA genome heterodimerization influences genome packaging and virion thermostability. Journal of Virology, 98(3), e01820-23.

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Single-cell RNA sequencing of pancreatic tumors

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Tumors and pancreas were enzymatically digested into a single-cell suspension precisely as we have previously described [10 ]. scRNAseq library generation was performed using the 10X Chromium System (10X Genomics Inc; Pleasanton, CA). Single cell suspensions were resuspended in PBS containing 0.04% w/v bovine serum albumin and brought to a concentration of 200–700 cells/μL. Appropriate volume of cells was loaded with single cell 5’ gel beads into a single cell chip and run on the Chromium Controller (10x Genomics, Inc; Pleasanton, CA). Dynabeads MyOne Silane magnetic beads (Thermo Fisher Scientific) were used to clean up the gel bead emulsion reaction mixture. Full-length, barcoded cDNA was amplified by PCR after cleanup. All samples were run on Agilent Tapestation 4200 using DNA high sensitivity D5000 tape. During library preparation, sample index and Illumina adapter sequences (Illumina Inc; San Diego, CA) were added. After library preparation quality control was performed using DNA D1000 tape on the Agilent Tapestation 4200 and final concentration was measured using the Qubit 4 Fluorometer DNA HS assay (Thermo Fisher Scientific). All samples were loaded at a concentration of 1.5 pM and run on the Illumina NextSeq500 High Output Flowcell. The run configuration was 26 base pairs (bp) × 98 bp × 8.

Hosein A.N., Dangol G., Okumura T., Roszik J., Rajapakshe K., Siemann M., Zaid M., Ghosh B., Monberg M., Guerrero P.A., Singhi A., Haymaker C.L., Clevers H., Abou-Elkacem L., Woermann S.M, & Maitra A. (2021). Loss of Rnf43 accelerates Kras-mediated neoplasia and remodels the tumor immune microenvironment in pancreatic adenocarcinoma. Gastroenterology, 162(4), 1303-1318.e18.

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