Ion xpress fragment library kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion Xpress™ Fragment Library kit is a DNA library preparation solution for next-generation sequencing applications. The kit provides the necessary reagents and protocols to generate fragment libraries from DNA samples. It includes enzymes, buffers, and adapters required for library construction, enabling users to prepare samples for downstream sequencing on compatible platforms.

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6 protocols using ion xpress fragment library kit

Genomic DNA Extraction and Sequencing of C. difficile Strains

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Genomic DNA was extracted from overnight TPGY cultures as described previously
[10 (link)] and further purified using the DNA Clean & Concentrator-5 kit (Zymo Research, Irvine, CA). Fragment libraries were constructed using 100 ng of genomic DNA with the Ion Xpress™ Fragment Library kit and size-selection was performed using the E-Gel® Agarose Gel Electrophoresis System (Life Technologies, Grand Island, NY). Library concentrations were determined using the Qubit® dsDNA HS Assay (Life Technologies, Grand Island, NY) and diluted to a final concentration of 18 pM. Templates were generated using the Ion OneTouch™ 200 Template Kit v2 and sequencing was performed using the Ion Torrent™ Personal Genome Machine (PGM™) with 314 v2 chips. Barcoded fragment libraries of various "picks" of strains CDC 52271 and CDC 52298 were generated with the Ion Torrent-compatible Bioo Scientific (Austin, TX) NEXflex™ DNA barcodes and sequenced with a 316 v2 chip.

Raphael B.H., Shirey T.B., Lúquez C, & Maslanka S.E. (2014). Distinguishing highly-related outbreak-associated Clostridium botulinum type A(B) strains. BMC Microbiology, 14, 192.

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Ion Xpress Library Preparation for Ion PGM Sequencing

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The Ion Xpress™ Fragment Library Kit (Life Technologies, Carlsbad, CA) was used to construct a library for shotgun sequencing on the Ion Personal Genome Machine (PGM, Ion Torrent/Life Technologies). The DNA was subjected to enzymatic fragmentation and library was constructed using the Ion Fragment Library Kit protocol. Adaptors were ligated to the repaired fragment ends followed by size selection. The library was PCR amplified using forward and reverse primers. The quality and the quantity of each of the libraries were assessed with the 2100 Bioanalyzer (DNA High Sensitivity Chip, Agilent Technologies, Sunnyvale, CA). Templates were then prepared and enriched on the Ion Sphere Particles™ (ISPs) using the Ion Xpress™ Template Kit (Life Technologies) and subjected to sequencing using the Ion Express Template 200 kit (Life Technologies, USA). Signal processing and base calling were performed with Torrent Analysis Suite version 3.4.1.

Singh K.M., Reddy B., Patel A.K., Panchasara H., Parmar N., Patel A.B., Shah T.M., Bhatt V.D, & Joshi C.G. (2014). Metagenomic analysis of buffalo rumen microbiome: Effect of roughage diet on Dormancy and Sporulation genes. Meta Gene, 2, 252-268.

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Ion Xpress Fragment Library Prep

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Using the Ion Xpress Fragment Library Kit, six unique barcode adapters (Table 2) were ligated
to the purified amplicons (Life Technologies Corp.; Cat. no. 4474009). Samples
were prepared following manufacturer protocol, except for one minor
modification, in which 1 µL of each adapter and barcode was used to avoid excess
that forms dimers. Following ligation and nick repair, samples were purified
using an ethanol solution and 1.4× volume of AMPure beads.

McDermid K.J., Kittle RP I.I.I., Veillet A., Plouviez S., Muehlstein L, & Balazs G.H. (2020). Identification of Gastrointestinal Microbiota in Hawaiian Green Turtles (Chelonia mydas). Evolutionary Bioinformatics Online, 16, 1176934320914603.

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Genomic DNA Extraction and Ion Torrent Sequencing

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Genomic DNA was extracted from the bacteriophages, as described by Irshad, Waqas & Saadia (2012) (link), with minor adjustments. The purity and concentration of the DNA was evaluated using a Nanodrop Bioanalyzer N1000 (Thermo Scientific, Waltham, MA, USA). In addition, sequencing libraries were prepared by shearing 1 µg of the phage DNA, to generate blunt-ended fragments, after which the Ion adapters were linked using an Ion Xpress™ Fragment Library Kit (Life Technologies, Carlsbad, CA, USA), in accordance with the manufacturer’s instructions. The produced fragments were amplified employing the Ion OneTouch 200 Template kit (Life Technologies, Carlsbad, CA, USA). Furthermore, libraries were sequenced on an Ion Torrent PGM semiconductor sequencer using the Ion Torrent 314 chip, by applying the standard protocol (Life Technologies, Carlsbad, CA, USA).

Sharaf A., Oborník M., Hammad A., El-Afifi S, & Marei E. (2018). Characterization and comparative genomic analysis of virulent and temperate Bacillus megaterium bacteriophages. PeerJ, 6, e5687.

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DNA Extraction and Metagenomic Sequencing

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Whole metagenomic DNA was extracted using the FastDNA Spin Kit for DNA Isolation (MP Biomedicals), eluted in water, and quantified using the Qubit High Sensitivity dsDNA Assay Kit as previously described (Mottawea et al., 2016 (link)).
Shotgun metagenomic sequencing libraries for both virome and metagenome DNA were prepared and barcoded using the Ion Xpress Fragment Library Kit and Ion Xpress Barcode Adapters (Thermo Fisher), with sonication performed on the Covaris S220 Ultra Sonicator following the manufacturer's instructions. Libraries were visualized with the High Sensitivity DNA Kit (Agilent) on the 2100 Bioanalyzer. Samples were templated and loaded on two Ion PI Chips (virome and metagenome samples on separate chips) by an Ion Chef using the Hi-Q Chef Kit and sequenced on an Ion Proton with the Hi-Q Sequencing 200 Kit following manufacturer's instructions.

Yan A., Butcher J., Mack D, & Stintzi A. (2020). Virome Sequencing of the Human Intestinal Mucosal–Luminal Interface. Frontiers in Cellular and Infection Microbiology, 10, 582187.

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Ion Proton Sequencing Library Preparation

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DNA was sheared with Ion Shear reagents and the Ion Xpress Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA) was used for subsequent library preparation. [AQ10] After ligation of the respective Ion Xpress barcode adapters, samples were amplified by 17 or 23 PCR cycles. Amplified libraries were quantified on Bioanalyzer DNA High-Sensitivity Chips (Agilent Technologies, Santa Clara, CA) and diluted to 9 pM. Emulsion PCR was performed on the Ion OneTouch System, followed by enrichment for template positive Ion Sphere Particles. Sequencing sample was loaded on an Ion Proton chip and sequenced on the Ion proton sequencer (Thermo Fisher Scientific) according to the manufacturer’s instructions.

Göbel U., Arce A.L., He F., Rico A., Schmitz G, & de Meaux J. (2018). Robustness of Transposable Element Regulation but No Genomic Shock Observed in Interspecific Arabidopsis Hybrids. Genome Biology and Evolution, 10(6), 1403-1415.

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