Ion ampliseq library kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion AmpliSeq Library Kit is a molecular biology tool designed for the preparation of amplicon libraries for targeted sequencing on Ion Torrent sequencing platforms. The kit provides a streamlined workflow for generating libraries from genomic DNA or cDNA samples.

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Ion AmpliSeq (43 citations) Ion AmpliSeq Library Kits 2.0 (10 citations)

32 protocols using ion ampliseq library kit

Targeted PIK3CA Sequencing Panel

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We designed an Ion AmpliSeq panel for targeted sequencing of the 21 coding exons of PIK3CA and ten nucleotides of all flanking introns (NM_006218.2) (http://www.ampliseq.com). Theoretical horizontal coverage was 96.75%, with 16 bp in the 5’UTR (exon 1) and 32 bp in exon 20 not covered. The panel consisted of 2 pools of primers for multiplexed PCR-amplification with Ion Ampliseq Library kit, and sequencing on an Ion Personal Genome Machine (PGM) or an ion Proton (Thermo Fisher Scientific). Reads were aligned to the human reference sequence hg19, using the Torrent Suite Server. Bam files were imported into Highlander software package (https://sites.uclouvain.be/highlander/) for analysis. We selected variants with at least 5 mutant reads representing at minimum 1% of all alleles by interrogating all positions reported with at least 4 changes in the COSMIC database (https://cancer.sanger.ac.uk/cosmic). Samples needed to have an average coverage above 500 × to be considered not to contain a mutation. Mutations with a VAF below 1% but confirmed by ddPCR when DNA was available, were considered to contain a mutation.

Brouillard P., Schlögel M.J., Homayun Sepehr N., Helaers R., Queisser A., Fastré E., Boutry S., Schmitz S., Clapuyt P., Hammer F., Dompmartin A., Weitz-Tuoretmaa A., Laranne J., Pasquesoone L., Vilain C., Boon L.M, & Vikkula M. (2021). Non-hotspot PIK3CA mutations are more frequent in CLOVES than in common or combined lymphatic malformations. Orphanet Journal of Rare Diseases, 16, 267.

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Exome Sequencing of Obesity Siblings

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In this study, we have sequenced exome of two siblings (one with early onset of obesity and other without any disease‐specific symptoms) and their parents. The initial amount of 100 ng of whole genomic DNA of each subject was amplified using Ampliseq RDY panel kit from Thermo Fisher Scientific. The panel encompasses ~294,000 primer pairs across 12 primer pools covering more than 97% of CCDS with 5 bp padding around exons. The library was prepared using Ion Ampliseq library kit plus (ThermoFisher scientific) and bar‐coded libraries were pooled in equimolar concentration to make the final concentration 500 ng. Library amplification and enrichment was done using Ion PI Hi‐Q OT2 200 kit (ThermoFisher scientific) on Ion OneTouch 2 and Ion OneTouch ES instruments, respectively. Libraries were then sequenced using Ion PI Hi‐Q sequencing 200 Kit on Ion Proton system according to the manufacturer's protocol.

Bhatt A., Purani C., Bhargava P., Patel K., Agarbattiwala T., Puvar A., Shah K., Joshi C.G., Dhamecha N., Prabhakar M, & Joshi M. (2019). Whole exome sequencing reveals novel LEPR frameshift mutation in severely obese children from Western India. Molecular Genetics & Genomic Medicine, 7(7), e00692.

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Targeted Cancer Gene Sequencing

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Library preparation was performed using the Ion AmpliSeq™ Library Kit (Thermo Fisher Scientific). The 207 amplicons were from 50 oncogenes and tumor suppressor genes (Additional file 1: Table S1). Sequencing was performed on an Ion Proton System using Ion PI™ Hi-Q™ Chef Kit (Thermo Fisher Scientific).

Wang C., Luo Q., Huang W., Zhang C., Liao H., Chen K, & Pan M. (2021). Correlation Between Circulating Tumor Cell DNA Genomic Alterations and Mesenchymal CTCs or CTC-Associated White Blood Cell Clusters in Hepatocellular Carcinoma. Frontiers in Oncology, 11, 686365.

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BRCA1/2 Variant Detection using Ion Sequencing

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The libraries were prepared using the Ion AmpliSeq Library Kit, the Ion AmpliSeq BRCA1/2 Panel, and the Ion Xpress Barcode Adapters Kit, according to the manufacturer's instructions (ThermoFisher Scientific). Sequencing was performed on the Ion Personal Genome Machine (PGM) using the Ion PGM Hi-Q Sequencing Kit and on an Ion S5 sequencer (ThermoFisher Scientific) using the Ion 520 & Ion 530 Kit-Chef. The raw data generated during sequencing was processed using Torrent Server Suite 4.2–5.2 (ThermoFisher Scientific) and the Variant Callerv 4.2–5.2 application. The results were viewed using Integrative Genomics Viewer (IGV; Broad Institute). Additionally, Torrent Server Suite 4.2 generated FASTQ files that were used for analysis by other methods, including the CLC Genomics Workbench, version 7.5.1 (Qiagen) and the Galaxy platform (www.usegalaxy.org). The wANNOVAR program (www.wannovar.usc.edu) was used to annotate the detected variants from Torrent Server Suite and Galaxy. Details are described in S1 Supplementary Methods.

Kowalik A., Siołek M., Kopczyński J., Krawiec K., Kalisz J., Zięba S., Kozak-Klonowska B., Wypiórkiewicz E., Furmańczyk J., Nowak-Ozimek E., Chłopek M., Macek P., Smok-Kalwat J, & Góźdź S. (2018). BRCA1 founder mutations and beyond in the Polish population: A single-institution BRCA1/2 next-generation sequencing study. PLoS ONE, 13(7), e0201086.

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High-depth Targeted Sequencing for CRC

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Targeted high-depth sequencing was performed for genomic areas comprising 100 genes using a custom-designed CRC panel (Table S1) [16 (link)]. From each sample, 10 ng of DNA were used for multiplexed PCR amplification with the Ion Ampliseq Library kit (ThermoFisher Scientific), using two amplicon pools per DNA sample. Samples were ligated to Ion Xpress Barcode Adapters (ThermoFisher Scientific) and purified using Agencourt AMPure beads (Beckman Coulter). All six samples were combined on a 316v2 chip and sequenced on an Ion Torrent PGM device (ThermoFisher) with an average read depth of 580–700.

Pfohl U., Loskutov J., Bashir S., Kühn R., Herter P., Templin M., Mamlouk S., Belanov S., Linnebacher M., Bürtin F., Vetter M., Reinhard C., Wedeken L, & Regenbrecht C.R. (2022). A RAS-Independent Biomarker Panel to Reliably Predict Response to MEK Inhibition in Colorectal Cancer. Cancers, 14(13), 3252.

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Targeted Amplicon Sequencing of Brain Metastasis

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Genomic DNA from brain metastasis biopsy tissue (case #1) was isolated using the DNeasy Blood & Tissue Kit (Qiagen) according to the manufacturer’s protocol. Ion AmpliSeq Colon and Lung Cancer Research Panel v2 (Thermo Fisher Scientific), a previously validated panel for targeted amplicon sequencing was utilized (Thermo Fisher Scientific) [20 (link)]. Briefly, 10 nanograms of gDNA was amplified by PCR using Ion AmpliSeq Library Kit (Thermo Fisher Scientific) and the sequencing was performed on an Ion PGM System according to the manufacturer’s protocol. Sequencing reads were multiplexed, quality-filtered, and aligned to the human reference genome (GRCh37) using the Torrent Suite software (ver. 5.0.4; Thermo Fisher Scientific). Variants were identified with the Variant Caller software (ver. 5.0.4.0; Thermo Fisher Scientific). The quality of all variants called was manually confirmed by IGV software (ver. 2.3.59).

Yoshida R., Sasaki T., Umekage Y., Tanno S., Ono Y., Ogata M., Chiba S., Mizukami Y, & Ohsaki Y. (2018). Highly sensitive detection of ALK resistance mutations in plasma using droplet digital PCR. BMC Cancer, 18, 1136.

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Fusion Gene Detection by Oncomine Assay

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Gene fusions were assessed on total RNA by Oncomine™ Comprehensive Assay Plus RNA panel (OCA Plus RNA, Thermo Fisher Scientific) that covers more than 1,300 isoforms across 49 known cancer-related fusion drivers. Libraries were prepared using the Ion AmpliSeq Library Kit plus with OCA RNA plus pools and sequenced on an Ion GeneStudio S5 Prime sequencer using Ion 530 chips, Ion 510 & Ion 520 & Ion 530 Kit-Chef, and Ion Chef System (Thermo Fisher Scientific), according to the manufacturer’s instructions. Data were processed by Torrent Suite™ and analyzed by Ion Reporter™ software (5.18 version) with the “Oncomine Comprehensive Plus - w2.2 - Fusions - Single Sample” workflow.

Minna E., Devecchi A., Pistore F., Paolini B., Mauro G., Penso D.A., Pagliardini S., Busico A., Pruneri G., De Cecco L., Borrello M.G., Sensi M, & Greco A. (2023). Genomic and transcriptomic analyses of thyroid cancers identify DICER1 somatic mutations in adult follicular-patterned RAS-like tumors. Frontiers in Endocrinology, 14, 1267499.

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Gene Panel Sequencing of Inflammatory Markers

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DNA was extracted from the buffy coat of blood samples using a QIAamp DNA maxi kit (Qiagen, Mississauga, ON, Canada). The gene panel studied included 16 genes; IL10, PCSK9, AMPD1, FCGR2A, HSP, MBP, LDLR, IL1, IL1R, IL1RN, HIF-1, CD14, TNF-alpha, IL6, TLR4, and SVEP1 (Table 1). Specific primers were designed using Ion AmpliSeq Designer (Life Technologies, USA), covering all exons and 100 base pair of exon intron junction. The exome library was prepared using the Ion AmpliSeq Library Kit (Thermo Fisher Scientific, USA) and sequenced using Ion PGM Hi-Q Sequencing Kit and ‘318’ sequencing chips on Ion Torrent PGM System (Thermo Fisher Scientific, USA). Analyses were performed using Torrent Suite and Ion Reporter (Thermo Fisher Scientific, USA). Sequence reads were aligned to the human GRC37/hg19.

Siddiqui S., Gurung R.L., Liu S., Ping Seet E.C, & Lim S.C. (2019). Genetic Polymorphisms and Cytokine Profile of Different Ethnicities inSeptic Shock Patients and their Association with Mortality. Indian Journal of Critical Care Medicine : Peer-reviewed, Official Publication of Indian Society of Critical Care Medicine, 23(3), 135-138.

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Ion Torrent PGM Ampliseq Sequencing

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We used 20ng of DNA for library preparation using the Ion Ampliseq Library kit (Thermo Fisher Scientific). Regions were targeted by primers distributed into two amplicon pools per DNA sample for the PanNEN panel and 4 amplicon pools per DNA sample for Comprehensive Cancer Panel (CCP). Together, the panels covered 432 genes. Upon ligation to Ion Xpress Barcode Adapters (Thermo Fisher Scientific) and purification using Agencourt AMPure beads (Beckman Coulter), two samples were mixed at equal ratio on a 318v2 sequencing chip. Using the Ion Torrent PGM (Thermo Fisher Scientific), the samples were sequenced at an average read depth of 1158 reads using the PanNEN panel and an average read depth of 217.03 reads for CCP, in order to generate the raw intensity data.

Simon T., Riemer P., Jarosch A., Detjen K., Di Domenico A., Bormann F., Menne A., Khouja S., Monjé N., Childs L.H., Lenze D., Leser U., Rossner F., Morkel M., Blüthgen N., Pavel M., Horst D., Capper D., Marinoni I., Perren A., Mamlouk S, & Sers C. (2022). DNA methylation reveals distinct cells of origin for pancreatic neuroendocrine carcinomas and pancreatic neuroendocrine tumors. Genome Medicine, 14, 24.

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SARS-CoV-2 Whole Genome Sequencing

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Complementary DNA (cDNA) was prepared from RNA elutes using the SuperScript VILO cDNA Synthesis kit (Invitrogen, Thermo Fisher Scientific, USA) as per the manufacturer’s instructions. Among 471 SARS-CoV-2 positive samples reported during the study period, whole genome sequencing was carried out on 66 of 92 samples with cycle threshold (Ct) values <25 as per the World Health Organization (WHO) criterion. Ion AmpliSeq library kit (ThermoFisher Scientific, Waltham, USA) was used for the library preparation, and the final library was adjusted to a final concentration of 75 pM using the low TE buffer and loaded onto Ion Chef instrument for emulsion PCR, enrichment, and subsequent placement onto an Ion 540 chip. Next-generation sequencing (NGS) was conducted using the Ion Torrent NGS System using the Ion GenStudio S5 Plus System (Thermo Fisher Scientific, Waltham, USA). Data analysis was performed using Torrent Suite^™ software ver.5.18.1. The consensus sequence was analysed using IRMA report ver.1.3.0.2. Annotation was performed using the SnpEff program. The reads were aligned to the Wuhan Hu-1 strain as a reference genome (NCBI ID: NC_045512.2).

Selvavinayagam S.T., Sankar S., Yong Y.K., Murugesan A., Suvaithenamudhan S., Hemashree K., Rajeshkumar M., Kumaresan A., Pandey R.P., Shanmugam S., Arthydevi P., Kumar M.S., Gopalan N., Kannan M., Cheedarla N., Tan H.Y., Zhang Y., Larsson M., Balakrishnan P., Velu V., Byrareddy S.N., Shankar E.M, & Raju S. (2024). Emergence of SARS-CoV-2 Omicron Variant JN.1 in Tamil Nadu, India - Clinical Characteristics and Novel Mutations. medRxiv.

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