Ion library equalizer kit

Manufactured by Thermo Fisher Scientific

Sourced in United States

The Ion Library Equalizer Kit is a laboratory equipment product designed to normalize the abundance of DNA library fragments prior to sequencing. It enables more balanced representation of individual sequences within a DNA library, helping to improve the quality and efficiency of subsequent sequencing analysis.

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19 protocols using ion library equalizer kit

Aneuploidy Analysis by NGS and aCGH

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The biopsied cells were whole genome amplified (WGA) using the Sureplex DNA Amplification System (Bluegnome) according to the manufacturer’s protocol. The WGA products were then processed for aneuploidy analysis by NGS or aCGH. Testing by aCGH was processed using Bluegnome 24sure V3 protocol (Illumina, Inc.) according to the manufacturer’s protocol. WGA products were fluorescently labeled with Cy3 and Cy5 dyes and random primers and subsequently were prepared to be hybridized to 24sure V3 array slides. Aneuploidy data analysis was performed using BlueFuse Multi Software. Testing by NGS was processed using Ion Torrent PGM (Ion Torrent) technology. Libraries were prepared by fragmenting WGA products with DNA concentrations of 100 ng using Ion Xpress Plus Fragment Library Kit (Life Technologies). Library fragments were selected at 200 bp using E-Gel SizeSelect Gels (Life Technologies) and then were normalized to 100 pM using an Ion Library Equalizer kit (Life Technologies). Libraries were subsequently pooled together into a mastermix and clonal amplified on the Ion One Touch 2 system. The template was then loaded into a 316 V2 chip (Life Technologies) and sequenced at 200 bp. Aneuploidy data analysis was performed using Ion Reporter software, using Low-Coverage Whole-Gnome workflow.

Coates A., Bankowski B.J., Kung A., Griffin D.K, & Munne S. (2016). Differences in pregnancy outcomes in donor egg frozen embryo transfer (FET) cycles following preimplantation genetic screening (PGS): a single center retrospective study. Journal of Assisted Reproduction and Genetics, 34(1), 71-78.

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Targeted Genomic Sequencing Protocol

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Capture of the target regions was performed with reagents from a custom design HaloPlex Target Enrichment kit (Agilent Technologies), according to the HaloPlex Target Enrichment System Protocol. Briefly, the protocol consisted of the following steps: (1) digestion of gDNA with restriction enzymes; (2) hybridization of fragments to probes whose ends are complementary to the target fragments (during this step, fragments are circularized and sequencing and barcode adapters are incorporated); (3) capture of target DNA using streptavidin beads and ligation of circularized fragments; and (4) PCR amplification of captured target libraries.
Quality control of all libraries was performed on the Agilent Bioanalyzer using a High Sensitivity chip. Template dilutions were calculated after library concentrations were normalized to ~100 pM using the Ion Library Equalizer kit (Life Technologies). Library templates were clonally amplified using the Ion One Touch 2^™, following the manufacturers’ protocol. Recovered template-positive ion sphere particles (ISPs) were subjected to enrichment according to the manufacturer’s protocol. Samples were subjected to the standard Ion PGM 200 Sequencing v2 protocol using Ion 318 v2 chips (Life Technologies). Up to three samples were loaded per Ion 318 v2 chip due to variable coverage uniformity.

Stoddard J.L., Niemela J.E., Fleisher T.A, & Rosenzweig S.D. (2014). Targeted NGS: A Cost-Effective Approach to Molecular Diagnosis of PIDs. Frontiers in Immunology, 5, 531.

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Ion Proton Sequencing Protocol

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The library was diluted to a concentration of 100 pM using the Ion Library Equalizer kit (Life Technologies, United States) before template preparation. Enriched, templated ion sphere particles were obtained using a DA8600 Ion Proton sequencer (DAANGENE, China) equipped with an Ion P1 chip (Life Technologies, United States).

Zeng H.H., Yang Z., Qiu Y.B., Bashir S., Li Y, & Xu M. (2022). Detection of a novel panel of 24 genes with high frequencies of mutation in gastric cancer based on next-generation sequencing. World Journal of Clinical Cases, 10(15), 4761-4775.

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Targeted Next-Gen Sequencing of Sinonasal Tumors

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Targeted next-generation sequencing was performed on DNA from 9 inverted sinonasal papillomas, 4 sinonasal squamous cell carcinomas associated with inverted papillomas, and 3 sinonasal squamous cell carcinomas not associated with a papilloma. Sequencing libraries were generated using Life Technologies’ Ion AmpliSeq Cancer Hotspot Panel v2. 10 ng of starting DNA from each sample block was amplified. Each library was barcoded (IonXpress Barcode Kit, Life Technologies) and equalized (Ion Library Equalizer Kit, Life Technologies) to a final concentration of approximately 100 pM. Emulsification PCR using combined barcoded libraries was performed using the One Touch 2 instrument. Template-positive Ion Sphere particles were then enriched using the One Touch ES instrument per the manufacturer's recommendations. Sequencing was performed on a 318v2 chip on the Ion Torrent PGM following the recommended protocol. Reads were aligned to hg19 and variants were called using the Torrent Suite 4.0.2 and Ion Reporter 4.0. Variants were assessed using the Broad Institute's Integrated Genomics Viewer (IGV 2.3). Candidate somatic variants from the Ion AmpliSeq Cancer Hotspot Panel were defined as those in regions with a depth greater than 100X and a variant frequency greater than 10%. Synonymous variants and variants reported in 1000 Genomes were excluded.

Udager A.M., Rolland D.C., McHugh J.B., Betz B.L., Murga-Zamalloa C., Carey T.E., Marentette L.J., Hermsen M.A., DuRoss K.E., Lim M.S., Elenitoba-Johnson K.S, & Brown N.A. (2015). High frequency targetable EGFR mutations in sinonasal squamous cell carcinomas arising from inverted sinonasal papilloma. Cancer research, 75(13), 2600-2606.

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Ion Ampliseq Cancer Hotspot Panel v2 Protocol

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The Ion Ampliseq Cancer Hotspot Panel v2 on the Ion Torrent platform was performed to detect 2790 mutations in 50 oncogenes and tumour suppressor genes^{16 (link),30 (link)}. In brief, 10 ng of genomic DNAs extracted from 55 frozen tumour samples were used to construct barcoded DNA libraries utilizing the Ion Ampliseq Library Kit 2.0 (Thermo Fisher Scientific). The obtained libraries were optimized using Ion Library Equalizer Kit (Thermo Fisher Scientific), and then sequenced using an Ion Personal Genome Machine or Ion S5XL platform (Thermo Fisher Scientific). The sequencing reads were aligned to the reference genome build hg19, GRCh37, and converted into BAM files using Ion Torrent Suite software (Thermo Fisher Scientific). Sequence variants were called using Ion Reporter 5.0 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The average sequencing depth reached at least 1500-fold per sample.

Watanabe T., Nanamiya H., Kojima M., Nomura S., Furukawa S., Soeda S., Tanaka D., Isogai T., Imai J.I., Watanabe S, & Fujimori K. (2020). Clinical implication of oncogenic somatic mutations in early-stage cervical cancer with radical hysterectomy. Scientific Reports, 10, 18734.

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Multiplex Library Preparation for NSCLC and mCRC

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For DNA libraries preparation, multiplex PCR was performed on 10 ng of DNA. After primer digestion and barcode ligation, library fragments were purified with Agencourt^® AMPure^® XP (Beckman Coulter, Brea, CA, USA). Finally, quantification and dilution (100 pM) of the amplified libraries was performed using the Ion Library Equalizer Kit (ThermoFisher Scientific) as described by the manufacturer.
RNA libraries preparation included a previous cDNA synthesis step from 10 ng of RNA using the SuperScript kit VILO cDNA synthesis kit (ThermoFisher Scientific). In this case, a multiplex PCR amplification of cDNA was performed. Library quantification was carried out by qPCR, inferring the concentration from a standard curve generated with Ion Library Quantification Kit (ThermoFisher Scientific). RNA libraries were diluted to a concentration of 100 pM.
In NSCLC, DNA and RNA libraries from eight patients were combined in a 4:1 proportion, generating the library pool. In mCRC samples, 10 DNA libraries were combined in equal proportion.

Simarro J., Murria R., Pérez-Simó G., Llop M., Mancheño N., Ramos D., de Juan I., Barragán E., Laiz B., Cases E., Ansótegui E., Gómez-Codina J., Aparicio J., Salvador C., Juan Ó, & Palanca S. (2019). Development, Implementation and Assessment of Molecular Diagnostics by Next Generation Sequencing in Personalized Treatment of Cancer: Experience of a Public Reference Healthcare Hospital. Cancers, 11(8), 1196.

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Routine Molecular Screening for Colon and Lung Cancer

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Routine molecular screening was performed at diagnosis for all patients on tumor biopsies. DNAs were extracted on a Maxwell 16 Forensic Instrument (Promega, France) using Maxwell 16 FFPE Plus LEV DNA Purification Kit (Promega, France) for FFPE samples. Quantification was done by Qubit dsDNA BR Assay Kit (Thermo Fisher Scientific, France). Colon and Lung Cancer Panel V2 libraries were prepared using the Ion Ampliseq library preparation kit v2 from 30 ng of tumor DNA. Libraries were normalized (Ion Library Equalizer Kit), pooled, processed on a Ion Chef System for template preparation and chip loading (Ion PI Hi-Q Chef Kit, Ion PI Chip Kit v3, Thermo Fisher Scientific), and sequenced on a Ion Proton System.

Giroux Leprieur E., Hélias-Rodzewicz Z., Takam Kamga P., Costantini A., Julie C., Corjon A., Dumenil C., Dumoulin J., Giraud V., Labrune S., Garinet S., Chinet T, & Emile J.F. (2020). Sequential ctDNA whole-exome sequencing in advanced lung adenocarcinoma with initial durable tumor response on immune checkpoint inhibitor and late progression. Journal for Immunotherapy of Cancer, 8(1), e000527.

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Ion Torrent PGM Sequencing Protocol

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A fragment library was generated with an input of 100 ng ds cDNA using the Ion Xpress Plus Fragment Library Kit (Thermo Scientific, U.S.A.). Next, DNA fragmentation, adaptor ligation, fragment size selection and library amplification were carried out according to manufacturer's instructions. The targeting of fragments of approximately 330 bp was performed with 1.5 × TBE 2% agarose gel (Helicon, Russia). Prior to emulsion PCR, the size distribution were assessed with 1.5 × TBE 2% agarose gel (Helicon, Russia); concentrations of the libraries were normalized with Ion Library Equalizer Kit (Thermo Scientific, USA). The fragment library was adjusted to approximately 26 pM and amplified with Ion Sphere particles™ (ISPs) by emulsion PCR using the Ion OneTouch™ Instrument with the Ion OneTouch™ 200 Template Kit (Thermo Scientific, USA) and template-positive ISP enrichment according to the manufacturer's protocol (Thermo Scientific, USA). About 50% of the ISPs were and then sequenced on an Ion 314™ chip using the Ion Torrent Personal Genome Machine (PGM™) (Thermo Scientific, USA) for 130 cycles (520 flows) with the Ion PGM™ 200 Sequencing Kit (Thermo Scientific, USA). Initial processing of ION PGM data carried out in an automatic mode using Ion Torrent Suite (Thermo Scientific, USA) software.

Pochtovyy A.A., Baranov O.Y., Rubel' I.E., Razumova O.A., Padutov V.E., Khromov A.V., Makhotenko A.V., Tkachuk A.P., Makarov V.V, & Gushchin V.A. (2017). Searching for active mobile genetic elements in dsRNA fraction of Pinus sylvestris having witches broom abnormalities. Genomics Data, 12, 102-108.

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Ion Torrent Targeted DNA Sequencing

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Genomic DNA was extracted from whole blood or saliva using the QIAGEN QIAamp DNA blood kit or tissue kit (QIAGEN Ltd., Germany). Custom amplification primers were designed to cover coding exons and flanking intron regions of the selected genes with Ion AmpliSeq Designer (Thermo Fisher Scientific, USA). Sample amplification and equalization were achieved using Ion AmpliSeq Library Kits 2.0 and the Ion Library Equalizer Kit, respectively (Thermo Fisher Scientific, USA). Amplified sequences were ligated with Ion Xpress Barcode Adapters (Thermo Fisher Scientific, USA). Emulsion PCR and subsequent enrichment were performed using the Ion OneTouch Template Kit v2.0 on Ion OneTouch 2 and Ion OneTouch ES, respectively (Thermo Fisher Scientific, USA). The final product was then sequenced on the Ion PGM sequencing platform (Thermo Fisher Scientific, USA). Raw data output from the sequencer was deposited in the DNA Data Bank of Japan (DDBJ) (http://www.ddbj.nig.ac.jp) under the accession number DRA004490, and uploaded to the Torrent Server (Life Technologies, USA) for variant calling, with NCBI GRCh37 as a reference. The resulting VCF files were analyzed by Ingenuity Variant Analysis (QIAGEN Ltd., Germany) for annotation and visualization.

Xing J., Kimura H., Wang C., Ishizuka K., Kushima I., Arioka Y., Yoshimi A., Nakamura Y., Shiino T., Oya-Ito T., Takasaki Y., Uno Y., Okada T., Iidaka T., Aleksic B., Mori D, & Ozaki N. (2016). Resequencing and Association Analysis of Six PSD-95-Related Genes as Possible Susceptibility Genes for Schizophrenia and Autism Spectrum Disorders. Scientific Reports, 6, 27491.

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Targeted NGS of Cancer Hotspot Mutations

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Genomic DNAs were extracted from fresh-frozen tissue samples using ISOGEN reagent (Nippongene, Tokyo, Japan), according to the manufacturer’s instructions. The quality and quantity of each DNA sample were assessed using NanoDrop One (ThermoFisher Scientific, Waltham, MA, USA).
The NGS for genomic DNAs from each sample was performed using the Ion Ampliseq Cancer Hotspot Panel v2, which covers approximately 2800 mutational hotspot regions from 50 cancer-related genes [12 (link), 13 (link)]. In brief, 10 ng of genomic DNAs extracted from 80 frozen tumor samples were used to construct barcoded DNA libraries utilizing an Ion Ampliseq Library Kit 2.0 (Thermo Fisher Scientific). The obtained libraries were optimized using an Ion Library Equalizer Kit (Thermo Fisher Scientific), and then sequenced using an Ion Personal Genome Machine or Ion S5XL platform (Thermo Fisher Scientific). The sequencing reads were aligned to the reference genome build hg19, GRCh37, and converted into BAM files using Ion Torrent Suite software (Thermo Fisher Scientific). Sequence variants were then called using Ion Reporter 5.0 (Thermo Fisher Scientific), according to the manufacturer’s instructions. The mean read depth of coverage in DNA sequencing was over 1500-fold.

Watanabe T., Nanamiya H., Endo Y., Kojima M., Nomura S., Furukawa S., Soeda S., Tamura H., Ryufuku M., Tanaka D., Isogai T., Imai J.I., Watanabe S, & Fujimori K. (2021). Identification and clinical significance of somatic oncogenic mutations in epithelial ovarian cancer. Journal of Ovarian Research, 14, 129.

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