For sequencing on the Ion PGM platform (ThermoFisher Scientific), libraries were prepared from 10 ng tumor DNA by using the Ion Torrent Ampliseq 2.0 kit (ThermoFisher Scientific) and CHPv2 panel according to the manufacturer's instructions. Samples were barcoded and quantified by qPCR using the Ion Xpress Barcode Adapter 1–96 kit and the Ion Library Taqman quantitation kit (ThermoFisher Scientific), respectively. Libraries from 12 samples were pooled at 20 pM concentrations for sequencing on the Ion PGM 318 v2 chip. Pooled libraries were clonally amplified on Ion Spheres (ThermoFisher Scientific). Emulsion PCR (e-PCR) was performed using the Ion PGM Template OT2 kit v2 and the Ion One Touch 2 system (ThermoFisher Scientific) as per manufacturer's instructions. Ion Sphere particles (ISPs) were enriched by using the Ion Torrent OneTouch ES (ThermoFisher Scientific). Enriched ISPs were loaded on a PGM 318 v2 chip and sequenced on the Ion PGM platform by using the Ion PGM sequencing kit (Fig 1 ).
Ion xpress barcode adapter 1 96 kit
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion Xpress Barcode Adapter 1–96 kit is a set of DNA barcode adapters designed for next-generation sequencing workflows. The kit contains 96 unique barcode sequences that can be used to label DNA samples, enabling sample multiplexing and pooling. The barcode adapters are intended to be ligated to DNA fragments prior to sequencing to facilitate sample identification and tracking.
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Lab products found in correlation
Ion library taqman quantitation kit, by Thermo Fisher Scientific (5 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (3 mentions)
Ion torrent ampliseq 2.0 kit, by Thermo Fisher Scientific (2 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (2 mentions)
Ion torrent systems proton system, by Thermo Fisher Scientific (2 mentions)
Ion pi hi q sequencing 200 kit, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp reagent, by Beckman Coulter (2 mentions)
Oncomine comprehensive assay v1, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq library kit, by Thermo Fisher Scientific (2 mentions)
Ion 540 chip kit, by Thermo Fisher Scientific (2 mentions)
Ion torrent s5xl machine platform, by Thermo Fisher Scientific (2 mentions)
Ion chef system, by Thermo Fisher Scientific (2 mentions)
Ion spheres, by Thermo Fisher Scientific (1 mentions)
Ion pgm platform, by Thermo Fisher Scientific (1 mentions)
Ion genestudio s5 system, by Thermo Fisher Scientific (1 mentions)
Ion ampliseq library kit plus, by Thermo Fisher Scientific (1 mentions)
7900ht fast real time pcr system, by Thermo Fisher Scientific (1 mentions)
Ion genestudio s5, by Thermo Fisher Scientific (1 mentions)
Ion hi q pi chip v3, by Thermo Fisher Scientific (1 mentions)
Ion 318 chip, by Thermo Fisher Scientific (1 mentions)
9 protocols using ion xpress barcode adapter 1 96 kit
1
Ion PGM Platform Sequencing of Tumor DNA
2
Targeted NGS Analysis of Thyroid Samples
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NGS analysis was performed on the Ion Gene Studio S5 system (Thermo Fisher Scientific) using two thyroid-specific custom panels, a DNA panel and an RNA panel, as previously described [21 (link), 22 (link)].
Briefly, two libraries were prepared from 15 ng of DNA and 10 ng of RNA with the Ion AmpliSeq™ Library Kit Plus using the IonXpress™ Barcode Adapter 1–96 Kit (Thermo Fisher Scientific). The purified libraries were quantified with Ion Library TaqMan® Quantitation Kit on the 7900HT Fast Real Time PCR system (Thermo Fisher Scientific). DNA and RNA pooled libraries were clonally amplified on the Ion One Touch2 System and sequenced on Ion Gene Studio S5 (Thermo Fisher Scientific) according to the manufacturer’s instructions and as previously described [23 (link)].
Data analysis was performed using Torrent Suite v.5.10 with Coverage Analysis and Variant Caller plugins and annotated with Ion Reporter 5.12 and the wANNOVAR web server. Gene fusion analysis was performed with Ion Reporter 5.12 software (Thermo Fisher Scientific) using the workflow for gene fusion detection [22 (link)].
Briefly, two libraries were prepared from 15 ng of DNA and 10 ng of RNA with the Ion AmpliSeq™ Library Kit Plus using the IonXpress™ Barcode Adapter 1–96 Kit (Thermo Fisher Scientific). The purified libraries were quantified with Ion Library TaqMan® Quantitation Kit on the 7900HT Fast Real Time PCR system (Thermo Fisher Scientific). DNA and RNA pooled libraries were clonally amplified on the Ion One Touch2 System and sequenced on Ion Gene Studio S5 (Thermo Fisher Scientific) according to the manufacturer’s instructions and as previously described [23 (link)].
Data analysis was performed using Torrent Suite v.5.10 with Coverage Analysis and Variant Caller plugins and annotated with Ion Reporter 5.12 and the wANNOVAR web server. Gene fusion analysis was performed with Ion Reporter 5.12 software (Thermo Fisher Scientific) using the workflow for gene fusion detection [22 (link)].
3
NGS-based Comprehensive Cancer Profiling
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10 ng DNA was used as a template to construct the amplicon libraries using Thermo Fisher scientific ION Proton(DA8600) platform. The custom-designed panel encompassed 60 cancer-related genes, including AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, GNAQ, IDH2, JAK1, JAK2, KRAS, MAP2K1, MET, MEK1, NOTCH1, NRAS, PIK3CA, PTEN, PIK3CA, SMAD4, STK11, and TP53 etc.
RNA-based NGS was conducted using a custom-designed panel which included probes spanning the MET exon 13-15 junction. Also, the fusions of ALK, RET, ROS1, and NTRK1 were detected. The libraries were prepared using multiple PCR capture method and Ion Torrent high-throughput sequencing. Then, the amplicon libraries were sequenced using an Ion Torrent Systems Proton system and a PI chip with barcoding performed using an Ion Xpress Barcode Adapter 1-96 Kit (Thermo Fisher Scientific, MA, USA).
RNA-based NGS was conducted using a custom-designed panel which included probes spanning the MET exon 13-15 junction. Also, the fusions of ALK, RET, ROS1, and NTRK1 were detected. The libraries were prepared using multiple PCR capture method and Ion Torrent high-throughput sequencing. Then, the amplicon libraries were sequenced using an Ion Torrent Systems Proton system and a PI chip with barcoding performed using an Ion Xpress Barcode Adapter 1-96 Kit (Thermo Fisher Scientific, MA, USA).
4
cfDNA-based Cancer Mutation Profiling
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10ng cfDNA was used to prepare libraries using the Ion Torrent Ampliseq 2.0 kit (ThermoFisher Scientific, Waltham, MA) and Ampliseq Cancer Hot Spot Panel v2 (CHPv2) according to the manufacturer's instructions. Samples were barcoded and quantified by qPCR using the Ion Xpress Barcode Adapter 1–96 kit and the Ion Library Taqman quantitation kit (ThermoFisher Scientific), respectively. Quantified libraries were pooled followed by e-PCR and sequenced on the Ion Proton using the Ion HI-Q PI Chip v3 and Ion PI HI-Q sequencing 200 kit (ThermoFisher Scientific) as described earlier [29 (link)].
Data analysis for NGS was performed as described earlier [29 (link)]. A cutoff of 300,000 reads with a quality score of AQ20 (one misaligned base per 100 bases) and a minimum sequencing depth of 250X was used as a measure of successful sequencing of a sample Sequencing results, mutations, and their respective allelic frequencies observed in cfDNA were compared with those identified in tumor tissue biopsy specimens to establish concordance.
Data analysis for NGS was performed as described earlier [29 (link)]. A cutoff of 300,000 reads with a quality score of AQ20 (one misaligned base per 100 bases) and a minimum sequencing depth of 250X was used as a measure of successful sequencing of a sample Sequencing results, mutations, and their respective allelic frequencies observed in cfDNA were compared with those identified in tumor tissue biopsy specimens to establish concordance.
5
Hearing Loss Genes Targeted Sequencing
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Amplicon libraries of the target exons were prepared with an Ion AmpliSeq™ Custom Panel (ThermoFisher Scientific, MA, USA) designed using Ion AmpliSeq™ Designer (https://www.ampliseq.com ) for 63 or 68 genes (listed in Table S1 ) (Kitano et al., 2017 ; Nishio et al., 2015 ) reported to cause nonsyndromic hearing loss (Hereditary Hearing loss Homepage; http://hereditaryhearingloss.org/ ) with the Ion AmpliSeq™ Library Kit 2.0 (ThermoFisher Scientific) and Ion Xpress™ Barcode Adapter 1‐96 Kit (ThermoFisher Scientific) according to the manufacturer's instructions. After the amplicon libraries were prepared, equal amounts of the libraries for six patients were pooled for one Ion PGM™ sequence reaction and those for 45 patients were pooled for one Ion Proton™ sequencing.
The emulsion PCR was performed with the Ion OneTouch™ System and Ion OneTouch 200 Template Kit v2 (ThermoFisher Scientific) or Ion PI™ Hi‐Q™ OT2 200 Kit according to the manufacturer's instructions. Sequencing was performed with an Ion torrent PGM™ system using the Ion PGM™ 200 Sequencing Kit and Ion 318™ Chip (ThermoFisher Scientific), or Ion Proton™ system using the Ion PI™ Hi‐Q™ Sequencing 200 Kit and Ion PI™ Chip (ThermoFisher Scientific) according to the manufacturer's instructions.
The emulsion PCR was performed with the Ion OneTouch™ System and Ion OneTouch 200 Template Kit v2 (ThermoFisher Scientific) or Ion PI™ Hi‐Q™ OT2 200 Kit according to the manufacturer's instructions. Sequencing was performed with an Ion torrent PGM™ system using the Ion PGM™ 200 Sequencing Kit and Ion 318™ Chip (ThermoFisher Scientific), or Ion Proton™ system using the Ion PI™ Hi‐Q™ Sequencing 200 Kit and Ion PI™ Chip (ThermoFisher Scientific) according to the manufacturer's instructions.
6
Ion Amplicon Library Preparation
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Amplicon libraries were constructed using the Ion AmpliSeq™ Library Kit Plus (Cat. No. A35907, Thermo Fisher Scientific), and the Ion Xpress™ Barcode Adapter 1–96 Kit (Cat. No. 4471250, Thermo Fisher Scientific) according to the manufacturer's instructions. Briefly, 10 ng of DNA were used to generate the sequencing libraries with two pools of 385 and 390 primers, respectively: after target amplification in 10 µl reactions, pool 1 and pool 2 amplification reactions were combined. Library preparation resulted in a single sample library, and each sample library was assigned a barcode adapter to differentiate between samples. Barcoded libraries were ran on high sensitivity D1000 Screen Tapes (Cat. No. 5067-5584, Agilent Technologies, Santa Clara, CA) using the Tapestation 2200 (Agilent Technologies) to assess both the quality and the band size, and calculate the final library pool molarity. Eight sample libraries were then normalized to a concentration of 100 pM before they were combined for subsequent template preparation.
7
Cancer Gene Panel Sequencing
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An equivalent of 10 ng DNA served as a template to construct the amplicon libraries using an Ion AmpliSeqTM Library Kit 2.0 (Thermo Fisher Scientific), followed by targeted NGS as reported previously.19 (link) The custom-designed panel encompassed 21 cancer-related genes, including AKT1, ALK, BRAF, CTNNB1, DDR2, EGFR, ERBB2, ERBB4, FGFR1, FGFR2, FGFR3, KRAS, MAP2K1, MET, NOTCH1, NRAS, PIK3CA, PTEN, SMAD4, STK11, and TP53 (Supplementary Table 1
RNA-based NGS was conducted using a custom-designed panel which included probes spanning the MET exon 13–15 junction. Also, fusions of ALK, RET, ROS1, and NTRK1 with other genes were detected. The libraries were prepared using the one-step PCR amplification method. Then, the amplicon libraries were sequenced on an Ion Torrent Systems Proton system and a PI chip with barcoding performed using an Ion Xpress Barcode Adapter 1–96 Kit (Thermo Fisher Scientific).
RNA-based NGS was conducted using a custom-designed panel which included probes spanning the MET exon 13–15 junction. Also, fusions of ALK, RET, ROS1, and NTRK1 with other genes were detected. The libraries were prepared using the one-step PCR amplification method. Then, the amplicon libraries were sequenced on an Ion Torrent Systems Proton system and a PI chip with barcoding performed using an Ion Xpress Barcode Adapter 1–96 Kit (Thermo Fisher Scientific).
8
Comprehensive Genomic Profiling using Oncomine Assay
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The Oncomine comprehensive assay v1 (Thermo Fisher Scientific), which examines 143 genes, was performed to detect single nucleotide variants (SNVs), copy number alterations (CNAs), insertions and deletions (indels), and fusions. DNA and RNA were amplified using the Ion AmpliSeq library kit 2.0 (Thermo Fisher Scientific). To prepare the barcoded library, the Ion Xpress Barcode Adapter 1–96 kit (Thermo Fisher Scientific) was combined with the non-barcoded adapter mix in the Ion AmpliSeq library kit. The resulting amplicons were purified using the Agencourt AMPure XP reagent (Beckman Coulter, Brea, CA, USA) and 70% ethanol, following the manufacturer’s instructions. The concentration of the final library was 50 pM. Libraries were quantified using the Ion Library TaqMan quantitation kit (Thermo Fisher Scientific). The final libraries were transferred to the Ion Chef System (Thermo Fisher Scientific) for automated template preparation. Sequencing was performed on the Ion Torrent S5XL Machine platform (Thermo Fisher Scientific) with an Ion 540 Chip kit (Thermo Fisher Scientific).
9
Comprehensive Oncomine DNA/RNA Sequencing
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We used the Oncomine comprehensive assay v1 (Thermo Fisher Scientific), which examines 143 oncogenes and tumor suppressor genes detecting single nucleotide variant (SNV), copy number alteration (CNA), indels, and fusions. Targeted DNA/RNA amplification of each tumor sample was performed using the Ion AmpliSeq Library Kit 2.0 (Thermo Fisher Scientific). For barcoded library preparation, the Ion Xpress Barcode Adapter 1–96 kit (Thermo Fisher Scientific) was substituted for the non-barcoded adapter mix in the Ion AmpliSeq Library Kit. The resulting amplicons were purified using Agencourt AMPure XP Reagent (Beckman Coulter, Brea, CA, USA) according to the manufacturer's instructions. The final library molecules were 50pM in concentration, which is appropriate for downstream template preparation. The libraries underwent quantification using the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific) according to the manufacturer's instructions. The final library molecules were 50pM in concentration, which is appropriate for downstream template preparation. The final libraries were transferred to the Ion Chef System (Thermo Fisher Scientific) for automated template preparation. Sequencing was performed on the Ion Torrent S5XL Machine platform (Thermo Fisher Scientific) with an Ion 540 Chip Kit (Thermo Fisher Scientific), according to the manufacturer's instructions.
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