Ion xpress barcode adapter kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion Xpress barcode adapter kit is a laboratory product designed to enable the addition of unique molecular identifiers, also known as barcodes, to DNA samples prior to next-generation sequencing. The kit provides the necessary reagents and protocols to facilitate this barcoding process, which is an important step in various genomic analysis workflows.

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6 protocols using ion xpress barcode adapter kit

Illumina and Ion Torrent Library Preparation for ITS Amplicons

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Illumina paired-end adapters with unique indexes were ligated to 100 ng of ITS1 amplicons using the TruSeq DNA Nano Sample Preparation Kit (Illumina). Library enrichment was performed with 10 cycles of PCR and purified using Agencourt Ampure Magnetic Beads (Beckman). Successful ligation of Illumina adapters results in 120 base pair shifts in the size of ITS amplicons and are confirmed using Agilent DNA 1000 Bioanalyzer assays (Supplementary Fig. 1C). Qubit fluorometric assay (Life Technologies) is used for final quantitation and libraries were then pooled at equimolar concentrations.
Afterwards, 100 ng of pooled ITS amplicons were used to generate Ion Torrent sequencing libraries using the Ion Xpress Library Kit (Life Technologies). Adapters and primers were diluted 1:10 to accommodate for the low input into the library preparation. Libraries were barcoded using Ion Xpress Barcode Adapter Kit (Life Technologies) to allow for multiplexed sequencing. Libraries were quantified and qualified in the same manner as the Illumina libraries.

Tang J., Iliev I.D., Brown J., Underhill D.M, & Funari V.A. (2015). Mycobiome: Approaches to Analysis of Intestinal Fungi. Journal of immunological methods, 421, 112-121.

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Comprehensive BRCA1/BRCA2 Exon Sequencing

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All coding exons and intron sequences of 20 bp around each exon were targeted in the BRCA1 and BRCA2 genes, ultimately resulting in a total size of 22 462 bp. Briefly, 30 ng of DNA (10 ng per pool) was amplified using the Ion AmpliSeq BRCA1 and BRCA2 Panel (Life Technologies) containing 167 primer pairs and an Ion AmpliSeq kit 2.0. The amplicons were ligated to adapters with barcodes of the Ion Xpress Barcode Adapter Kit (Life Technologies). The libraries with adapters were pooled for multiplexing. The amplicons were clonally amplified through emulsion PCR by using a IT OneTouch Template Kit 2.0 on an IT OneTouch system (Life Technologies) following the manufacturer's instructions. Template‐positive Ion Sphere Particles (ISPs) were enriched, and purified ISPs were loaded on an Ion 316 or 318 Chip. Twelve barcoded samples on an Ion 316 Chip and 32 barcoded samples on a 318 Chip were sequenced. Targeted sequencing was performed using the Ion PGM platform using an Ion PGM sequencing 200 kit following the manufacturer's instructions.

Paik E.S., Heo E.J., Choi C.H., Kim J., Kim J., Kim Y., Park S., Lee J., Kim J, & Kim B. (2021). Prevalence and clinical characterization of BRCA1 and BRCA2 mutations in Korean patients with epithelial ovarian cancer. Cancer Science, 112(12), 5055-5067.

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Targeted Deep Sequencing of CRC Samples

Cited 2 times

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We performed target deep sequencing of the genomic DNA extracted from the three CRC tissue samples and matched normal samples using a custom NGS panel (OncoChase-AS01, ConnectaGen, Seoul, Korea), targeting 95 cancer-related genes as described elsewhere [10 (link),11 (link)]. Tumor DNA was amplified, digested, and barcoded using the Ion Ampliseq library kit 2.0 (Thermo Fisher Scientific) and Ion Xpress barcode adapter kit (Thermo Fisher Scientific) as described elsewhere [10 (link)]. The libraries were then templated on an Ion Chef system (Thermo Fisher Scientific) using Ion 520 and Ion 530 Chef reagents (Thermo Fisher Scientific). The prepared libraries were sequenced on an Ion S5 sequencer using an Ion 530 chip and Ion S5 sequencing reagents (Thermo Fisher Scientific) as described elsewhere [10 (link)].

Min S., Shin S, & Chung Y.J. (2019). Detection of KRAS mutations in plasma cell-free DNA of colorectal cancer patients and comparison with cancer panel data for tissue samples of the same cancers. Genomics & Informatics, 17(4), e42.

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Next-Generation Sequencing of CPN1 Variants

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Genomic DNA was isolated using MagNA Pure (Roche, Meylan, France). DNA samples from probands of families B and C were analyzed by next-generation sequencing (Ampliseq custom panel, Thermo Scientific, Waltham, Mass), as described²⁶ (link) (see Table E1 in this article’s Online Repository at www.jaci-global.org for the genes submitted to the analysis). Briefly, DNA libraries were constructed for each sample using Ion AmpliSeq Library Kit 2.0 (Thermo Scientific) and indexed with a unique adapter using the Ion Xpress barcode adapter kit (Thermo Scientific). Template preparation, enrichment, and chip loading were carried out on an Ion Chef system (Thermo Scientific). Sequencing was performed on S5XL on 520 and 530 chips, using the Ion 510, Ion 520, and Ion 530 Kit-Chef workflow. Primary data were analyzed using the Ion Reporter software (Thermo Scientific). Sanger sequencing of exon 3 (CPN1ex3_F: 5′-AGTATTCAATCTGAAACCTTCATTTTT-3′, CPN1ex3_R: 5′-AGATGGCTTAGCAGTCTTTCTG-3′) was used to confirm CPN1 variants and to sequence DNA samples.

Vincent D., Parsopoulou F., Martin L., Gaboriaud C., Demongeot J., Loules G., Fischer S., Cichon S., Germenis A.E., Ghannam A, & Drouet C. (2024). Hereditary angioedema with normal C1 inhibitor associated with carboxypeptidase N deficiency. The Journal of Allergy and Clinical Immunology: Global, 3(2), 100223.

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Targeted NGS for Cancer-Related Genes

Cited 1 time

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We used a custom NGS panel, OncoChase-AS01 (ConnectaGen, Seoul, Korea), targeting 95 cancer-related genes (Supplementary Fig. 1) [10 (link)]. Ten nanograms of DNA was amplified, digested, and barcoded using the Ion Ampliseq Library kit 2.0 (Thermo Fisher Scientific) and Ion Xpress barcode adapter kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. The amplified libraries were quantified using a Qubit fluorometer, the Qubit dsDNA HS assay kit, and the Ion Library TaqMan Quantitation kit (Thermo Fisher Scientific). The libraries were then templated on an Ion Chef System (Thermo Fisher Scientific) using Ion 520 and Ion 530 Chef Reagents (Thermo Fisher Scientific) according to the manufacturer’s instructions. The prepared libraries were sequenced on an Ion S5 Sequencer using an Ion 530 chip and Ion S5 Sequencing Reagents (Thermo Fisher Scientific).

Yu S.M., Jung S.H, & Chung Y.J. (2018). Comparison of the Genetic Alterations between Primary Colorectal Cancers and Their Corresponding Patient-Derived Xenograft Tissues. Genomics & Informatics, 16(2), 30-35.

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Quantitative RNA Expression Analysis by NGS

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Quantitative RNA expression analysis was performed by amplicon-based next-generation sequencing (NGS) using Ion Torrent technology (Thermo Fisher Scientific, Waltham, MA, USA). The Ion AmpliSeq™ RNA Library Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used to construct the libraries from 20 ng of RNA. RNA was reverse transcribed, and targeted regions of RNA were PCR amplified using the Ion AmpliSeq™ RNA Cancer Panel (Thermo Fisher Scientific, Waltham, MA, USA) consisting of specific primers sets to amplify 50 target genes. (Additional file 1: Table S1) The Amplicons were then partially digested, and barcode adapters were ligated with the Ion Xpress™ Barcode Adapter Kit (Thermo Fisher Scientific, Waltham, MA, USA) to yield a barcoded library. Library concentrations were measured using the Qubit 2.0 Fluorometer (Thermo Fisher Scientific, Waltham, MA, USA).
Templates for sequencing were prepared using the Ion PGM™ Hi-Q™ OT2 Kit (Thermo Fisher Scientific) and Ion OneTouch™ 2 System (Thermo Fisher Scientific, Waltham, MA, USA). Ion Sphere™ particles were enriched with Ion OneTouch ES (Thermo Fisher Scientific, Waltham, MA, USA) and loaded onto an Ion 318™ Chip (Thermo Fisher Scientific, Waltham, MA, USA).
Sequencing was performed on the Ion Torrent PGM™ System using the Ion PGM™ Hi-Q™ Sequencing Kit (Thermo Fisher Scientific, Waltham, MA, USA).

Veija T., Koljonen V., Bohling T., Kero M., Knuutila S, & Sarhadi V.K. (2017). Aberrant expression of ALK and EZH2 in Merkel cell carcinoma. BMC Cancer, 17, 236.

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