Ion xpress barcode adapter 1 16 kit

Manufactured by Thermo Fisher Scientific

The Ion Xpress Barcode Adapter 1–16 kit is a set of pre-designed and validated adapters used for indexing DNA samples during library preparation for Ion Torrent sequencing systems. The kit contains 16 unique barcode sequences that can be ligated to DNA fragments, enabling the pooling of multiple samples in a single sequencing run.

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Ion Xpress Barcode Adapters 1–16 Kit (21 citations) Ion Xpress Barcode (10 citations)

6 protocols using ion xpress barcode adapter 1 16 kit

Fecal DNA Extraction and 16S Sequencing

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DNA was extracted from frozen fecal samples using the DNA stool Mini kit (Qiagen), following the manufacturer’s instructions. DNA concentrations were measured using QuBit 2.0 Fluorometer kit (Life Technologies). Bacterial 16S ribosomal RNA (rRNA) genes contain nine “hypervariable regions” (named from V1 to V9) that demonstrate considerable sequence diversity among different bacteria. These regions of bacterial 16S rRNA genes were amplified by PCR using two sets of primers V2-4-8 and V3-6,7–9 available in the Ion 16S Metagenomics kit (Life Technologies). The PCR products were purified using SPRI method (Solid Phase Reversible Immobilization) (Agencourt AMPURE XP). Each amplicon was then assessed for fragment size distribution and DNA concentration using a Bioanalyser 2100 (Agilent Technologies, USA). Library preparation followed the Ion Plus Fragment Library (Life Technologies). Products were first end-repaired and purified using SPRI method (Agencourt AMPURE XP). Then, library was bar-coded using the Ion Xpress Barcode Adapter 1–16 kit (Life Technologies) and sample was again purified using SPRI method. Finally, emulsion PCR and enrichment steps were carried out using the Ion PGM (Life Technologies). Sequencing was undertaken using 316 chips and Ion PGMSequencing 400 on the Ion Torrent Personal Genome Machine (PGM).

Mombo I.M., Berthet N., Lukashev A.N., Bleicker T., Brünink S., Léger L., Atencia R., Cox D., Bouchier C., Durand P., Arnathau C., Brazier L., Fair J.N., Schneider B.S., Drexler J.F., Prugnolle F., Drosten C., Renaud F., Leroy E.M, & Rougeron V. (2015). First Detection of an Enterovirus C99 in a Captive Chimpanzee with Acute Flaccid Paralysis, from the Tchimpounga Chimpanzee Rehabilitation Center, Republic of Congo. PLoS ONE, 10(8), e0136700.

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Targeted Panel Sequencing of Cardiomyopathy Genes

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Sixty-four candidate genes reported to be causative of inherited cardiomyopathy, according to Online Mendelian Inheritance in Man (http://omim.org) and PubMed literature retrieval, were selected for panel design. The gene list is shown in Table S1. The primers of overlapping amplicons covering the coding sequence region and flanking sequences (padding +25 base pairs) of each targeted gene were automatically generated by Ion AmpliSeq designer software. This design produced 3647 amplicons, which were divided into 2 primer pools (Life Technologies; Thermo Fisher Scientific). Each amplicon is about 250 base pairs. Libraries were constructed using the Ion AmpliSeq Library Kit v2.0 (Life Technologies; Thermo Fisher Scientific) and custom designed primer pools, according to the manufacturer’s instructions. DNA fragments from different samples were ligated with bar-coded sequencing adaptors using the Ion Xpress Barcode Adapter 1-16 Kit (Life Technologies; Thermo Fisher Scientific). The library was quantified with the Qubit 2.0 fluorometer (Invitrogen; Thermo Fisher Scientific).

Wu W., Lu C.X., Wang Y.N., Liu F., Chen W., Liu Y.T., Han Y.C., Cao J., Zhang S.Y, & Zhang X. (2015). Novel Phenotype–Genotype Correlations of Restrictive Cardiomyopathy With Myosin-Binding Protein C (MYBPC3) Gene Mutations Tested by Next-Generation Sequencing. Journal of the American Heart Association: Cardiovascular and Cerebrovascular Disease, 4(7), e001879.

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Ion AmpliSeq Library Preparation and Sequencing

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The library was constructed using Ion AmpliSeq™ (CCP) Library Kit 2.0 (Life Technologies) and Ion Xpress™ Barcode Adapter 1–16 Kit (Life Technologies) according to manufacturer’s instructions. Library quantification was done using the Ion Library TaqMan Quantitation Kit (Life Technologies) following standard procedure available. The qualified library was sequenced by the use of Ion S5XL Semiconductor Sequencer following the manufacturer’s user guide.

Almuzzaini B., Alghamdi J., Alomani A., AlGhamdi S., Alsharm A.A., Alshieban S., Sayed A., Alhejaily A.G., Aljaser F.S., Abudawood M., Almajed F., Samman A., Balwi M.A, & Aziz M.A. (2021). Identification of Novel Mutations in Colorectal Cancer Patients Using AmpliSeq Comprehensive Cancer Panel. Journal of Personalized Medicine, 11(6), 535.

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Ion Torrent Targeted Sequencing

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After slpH-specific PCR, amplicons were purified using the Qiaquick PCR Purification kit (Qiagen, Hombrechtikon, Switzerland). Purified amplicons were fragmented by sonication of 1 μg of DNA in 130 μL nuclease-free water at 50 W, 200 cycles/burst, and 20°C for 90 s on a Covaris M220 instrument (Covaris, Brighton, U.K.). The fragmented DNA was end-repaired and adapter-ligated using the Ion Xpress Plus Fragment Library Kit (Thermo Fisher Scientific) according to the manufacturer's instruction. Samples were barcoded using the Ion Xpress Barcode Adapter 1-16 kit (Thermo Fisher Scientific). After barcoding, the DNA was size-selected for 400 bp reads using an E-Gel® SizeSelect™ Agarose Gel (Thermo Fisher Scientific). The libraries were quantified by qPCR using the Ion Library Quantitation Kit (Thermo Fisher Scientific). Before preparing the template-positive ion sphere particles with the Ion PGM Hi-Q OT2 kit (Thermo Fisher Scientific), each library was diluted to 100 pM in 10 mM Tris-HCl (pH 8.0) and 0.1 mM EDTA, before being pooled. The sequencing was performed using either an Ion 314™ or 316™ chip and the Ion PGM Hi-Q Sequencing kit on an Ion Torrent sequencer (Thermo Fisher Scientific).

Moser A., Wüthrich D., Bruggmann R., Eugster-Meier E., Meile L, & Irmler S. (2017). Amplicon Sequencing of the slpH Locus Permits Culture-Independent Strain Typing of Lactobacillus helveticus in Dairy Products. Frontiers in Microbiology, 8, 1380.

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Quantifying L. helveticus Populations

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To determine L. helveticus populations, the culture-independent method described by Moser et al. (2017b (link)) was performed. Briefly, an approximately 1,000-bp region of the slpH gene was amplified by PCR and the derived amplicons were fragmented by sonication. The fragments were barcoded using the Ion Xpress Barcode Adapter 1–16 kit (Thermo Fisher Scientific), and the barcoded libraries were sequenced on an Ion 316^TM chip and the Ion PGM Hi-Q View Sequencing kit on an Ion Torrent sequencer (Thermo Fisher Scientific). On each chip, 16 barcoded libraries were sequenced.

Moser A., Schafroth K., Meile L., Egger L., Badertscher R, & Irmler S. (2018). Population Dynamics of Lactobacillus helveticus in Swiss Gruyère-Type Cheese Manufactured With Natural Whey Cultures. Frontiers in Microbiology, 9, 637.

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Ion Torrent Sequencing of Germline Mutations

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Using BR Qubit 3 fluorometer, high quality samples were used for the library preparation. Library preparation and barcoding were performed using an Ion Xpress Barcode Adapter 1–16 Kit (Thermo Fischer Scientific). The samples were normalized using Ion Library Equalizer™ (Thermo Fischer Scientific) following manufacturer protocol. The amplified library was quantified using the 7900HT qPCR platform (Thermo Fischer Scientific), and purified using ion chef following manufacturer protocol. Following the standard manufacturer instructions, the samples were sequenced on an Ion Torrent Personal Genome Machine (PGM) sequencer (ThermoFisher Scientific). To identify the germline mutations in our target samples, the Ion 316 chips v2 having 100X coverage was used.

Alfadhel M., Umair M., Almuzzaini B., Alsaif S., AlMohaimeed S.A., Almashary M.A., Alharbi W., Alayyar L., Alasiri A., Ballow M., AlAbdulrahman A., Alaujan M., Nashabat M., Al‐Odaib A., Altwaijri W., Al‐Rumayyan A., Alrifai M.T., Alfares A., AlBalwi M, & Tabarki B. (2019). Targeted SLC19A3 gene sequencing of 3000 Saudi newborn: a pilot study toward newborn screening. Annals of Clinical and Translational Neurology, 6(10), 2097-2103.

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