Ampure xp system

Manufactured by Thermo Fisher Scientific

The AMPure® XP system is a magnetic bead-based nucleic acid purification product. It is designed to facilitate the purification of DNA, RNA, and other biomolecules from complex samples.

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AMPure XP (2 citations) AMPure XP PCR purification system (2 citations)

3 protocols using ampure xp system

Ion Torrent PGM Sequencing Protocol

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Library preparation was performed by amplifying 10 ng genomic DNA, using the Ion AmpliSeq™ Library kit 2.0 (Thermo Fisher Scientific, Inc.) according to the manufacturer's protocol. Libraries were purified using the Agencourt^® AMPure^® XP system and quantified using the Qubit^® dsDNA HS assay kit (Invitrogen; Thermo Fisher Scientific, Inc.) and clonally amplified by emulsion PCR using the Ion OneTouch™ 2 system (Ion PGM™ Template Hi-Q™ view OT2 200 kit; Thermo Fisher Scientific, Inc.) all according to the manufacturer's protocol. The spheres were loaded on to a 316™ v2 chip and sequenced on the Ion Torrent PGM, using the Ion PGM™ Hi-Q™ view Sequencing 200 kit v2. Post-run analysis was performed using Torrent Suite™ version 5.0.4 (Thermo Fisher Scientific, Inc.). Coverage assessment was performed using the ‘coverage Analysis’ plug-in which provides information regarding the amplicons read coverage, and variants were called using the ‘variant Caller’ plug-in (12 (link)).

Zhao X., Shang X., Chen C., Liu L., Wang C., Zhao G., Zhang J, & Kong X. (2020). Identification of four novel mutations in the COL4A5 gene identified in Chinese patients with X-linked Alport syndrome. Biomedical Reports, 13(2), 4.

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Targeted Sequencing of COL4A Genes

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The library preparation was performed by amplifying 10 ng of genomic DNA, using the Ion AmpliSeq™ Library Kit 2.0 (Life Technologies). This kit allowed obtaining a barcoded library of the 194 amplicons, corresponding to the 151 exons of COL4A3/COL4A4/COL4A5 genes compatible with the Ion PGM platform, according to the Life Technologies protocol. Libraries were purified using Agencourt^® AMPure^® XP system and quantified using the Qubit^® dsDNA HS Assay Kit reagent (Invitrogen Corporation, Life Technologies, Carlsbad, CA), pooled at an equimolar ratio, annealed to carrier spheres (Ion Sphere™ Particles, Life Technologies) and clonally amplified by emulsion PCR (emPCR) using the Ion OneTouch™ 2 system (Ion PGM™ Template Hi‐Q™ view OT2 200 kit, Life Technologies). The spheres, carrying single‐stranded DNA templates, were loaded to 316™v2 chip and sequenced on the Ion Torrent PGM, using the Ion PGM™ Hi‐Q™ view Sequencing 200 kit v2, according to the protocol of Life Technologies. Postrun analysis was conducted using the latest version (v5.0.4) of the data analysis software Torrent Suite™ (Life Technologies). Coverage assessment was performed using the “coverage Analysis” plug‐in (v5.0.4) that gives information about the amplicons read coverage and variants were called using the “variant Caller” plug‐in (5.0.4).

Zhao X., Chen C., Wei Y., Zhao G., Liu L., Wang C., Zhang J, & Kong X. (2019). Novel mutations of COL4A3, COL4A4, and COL4A5 genes in Chinese patients with Alport Syndrome using next generation sequence technique. Molecular Genetics & Genomic Medicine, 7(6), e653.

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Ion AmpliSeq Library Prep and Sequencing

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The Ion AmpliSeq 2.0 Library Kit (Life Technologies, Carlsbad, CA) was used for library preparation. The kit allowed us to obtain a barcoded library of 184 amplicons, corresponding to the 151 exons col COL4A3/COL4A4/COL4A5 genes compatible with the ION S5 platform, according to the manufacturer’s protocol (https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0006735_AmpliSeq_DNA_RNA_LibPrep_UG.pdf).
Libraries were purified using Agencourt AMPure XP system and quantified using the Qubit^® dsDNA HS Assay Kit reagent (Invitrogen Corporation, Life Technologies), pooled at an equimolar ratio, annealed to carrier spheres (Ion Sphere Particles, Life Technologies) and clonally amplified by emulsion PCR (emPCR) using the Ion Chef system (Ion Chef, Life Technologies). Ion 510, 520, or 530 chips were loaded with the spheres carrying single stranded DNA templates and sequenced on the Ion Torrent S5 using the Ion S5 Sequencing kit, according to the manufacturer’s protocol.
Post-experiment analysis was conducted using the latest version (v5.8.0) of the data analysis software Torrent Suite (Life Technologies). A coverage assessment was performed using the ‘coverageAnalysis’ plug-in (v5.8.0.8) that gives information about the amplicon read coverage. Variants were called using the ‘variantCaller’ plug-in (v5.8.0.19).

Daga S., Donati F., Capitani K., Croci S., Tita R., Giliberti A., Valentino F., Benetti E., Fallerini C., Niccheri F., Baldassarri M., Mencarelli M.A., Frullanti E., Furini S., Conticello S.G., Renieri A, & Pinto A.M. (2019). New frontiers to cure Alport syndrome: COL4A3 and COL4A5 gene editing in podocyte-lineage cells. European Journal of Human Genetics, 28(4), 480-490.

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