The library for DNA sequencing using NGS was generated using the Fluidigm Access Array microfluidic chip as previously described38 (link). The confirmed target-specific ACE2 primers were then tagged with Fluidigm specific tag sequences and used for targeted next-generation sequencing38 (link). The prepared amplicon libraries were purified using AMPure XP beads (Beckman Coulter, USA) and quantified using a High Sensitivity DNA assay kit on BioAnalyzer (Agilent, USA). The libraries were further diluted to 1 pg for direct input into the emulsion polymerase chain reaction (PCR) with Ion SphereTM particles using the Ion Template OT2 kit (Ion OneTouch™ instrument). Enrichment of the clonal beads was carried out using the Ion OneTouch™ ES system following the manufacturer’s instructions (Thermo Fischer, USA). The pooled libraries were then sequenced using the Ion 520™ Chip on the Ion S5 XL Semiconductor sequencer, following the manufacturer’s instructions (Thermo Fisher).
Ion 520 chip
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion 520 Chip is a semiconductor-based sequencing chip designed for use with the Ion Torrent Sequencing System. It is capable of generating high-throughput DNA sequencing data. The core function of the Ion 520 Chip is to enable massively parallel sequencing of nucleic acid samples.
Automatically generated - may contain errors
Lab products found in correlation
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (13 mentions)
Ion s5 system, by Thermo Fisher Scientific (3 mentions)
Ion onetouch es system, by Thermo Fisher Scientific (2 mentions)
Ion xpress barcode adapters kit, by Thermo Fisher Scientific (2 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (2 mentions)
Dneasy blood kit, by Qiagen (2 mentions)
Ion torrent s5, by Thermo Fisher Scientific (2 mentions)
2100 bioanalyzer, by Agilent Technologies (2 mentions)
Ion 530 kit ot2, by Thermo Fisher Scientific (2 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (2 mentions)
Ion s5 xl sequencer, by Thermo Fisher Scientific (2 mentions)
Ion 520 kit chef kit, by Thermo Fisher Scientific (2 mentions)
Ion s5 xl semiconductor sequencer, by Thermo Fisher Scientific (1 mentions)
Ampure xp bead, by Beckman Coulter (1 mentions)
Bioanalyzer, by Agilent Technologies (1 mentions)
Ion s5 instrument, by Thermo Fisher Scientific (1 mentions)
Ion chef instrument, by Thermo Fisher Scientific (1 mentions)
Ion genestudio s5 system, by Thermo Fisher Scientific (1 mentions)
E gel safe imager, by Thermo Fisher Scientific (1 mentions)
E gel ibase, by Thermo Fisher Scientific (1 mentions)
15 protocols using ion 520 chip
1
NGS-based ACE2 Amplicon Sequencing
2
Liquid Biopsy Variant Analysis Pipeline
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For the template preparation, 4 libraries (diluted to 50 pM) were multiplexed, and sequencing was subsequently performed using Ion 520™ chip (5 × 106 of reads capability), loaded on Ion Chef™ Instrument and run on Ion S5 instrument (ThermoFisher). The sequencing reads were aligned and mapped to the reference human genome sequence (hg19) using the Torrent Mapping Alignment Program (TMAP). The plugin Torrent Variant Caller (TVC, version 5.8, ThermoFisher) with specific parameters for liquid biopsy inside the JSON (JavaScript Object Notation) file was run in order to detect and report only the variants hotspot alleles that meet criteria for calling, i.e. a call was made when at least two family tags shared an identical mutation (corresponding to two independent single mutated DNA alleles) and each family tag displayed at least 3 reads. Optimal results were obtained with Median Read Coverage > 25,000 and Median Molecular Coverage > 2,500 (Oncomine™ cfDNA Assays Part III: Variant Analysis User guide).
Review of all the hotspots calls was performed by uploading each Variant Call Format (VCF) file on IGV (Integrative Genomics Viewer, Cambridge MA,https://software.broadinstitute.org/software/igv/home ).
Review of all the hotspots calls was performed by uploading each Variant Call Format (VCF) file on IGV (Integrative Genomics Viewer, Cambridge MA,
3
Whole-Genome Amplification and NGS Analysis of Trophectoderm Biopsies
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Whole-genome amplification (WGA) on each biopsy was performed using the Rubicon PicoPLEX WGA kit (Agilent, CA, USA) as per the manufacturer's recommendations as detailed previously.[39 40 ] Following WGA, NGS of the trophectodermal biopsies was carried out. For constructing WGA library, Ion Xpress Plus fragment library kit and Ion Xpress barcode adapters kit were used as per the manufacturer's instructions (Thermo Fisher Scientific, MA, USA). Briefly, 150 ng of WGA DNA was fragmented to generate 280 base pair fragments. The purified fragmented DNA was barcoded and amplified as per the manufacturer's instructions (Thermo Fisher Scientific, MA, USA). These (a pool of 24 samples) were then loaded on an Ion 520 Chip and were sequenced on the Ion Gene Studio S5 system (Ion S5 System User Guide, Thermo Fisher Scientific, MA, USA). Approximately 4.8–5 million reads were obtained for each run generating 200,000–300,000 reads/embryo.
4
Whole-Genome Copy Number Alteration Profiling
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The presence of CNA was investigated with a whole-genome low-coverage NGS approach by using the Ampli1™ LowPass Kit for the Ion Torrent (Menarini Silicon Biosystems) workflow described by Ferrarini et al. [37 (link)]. The size selection of the pooled libraries was performed using E-Gel™ SizeSelect™ II Agarose Gels, 2% (Invitrogen, Carlsbad, CA, USA) on E-Gel™ iBase™ and E-Gel™ Safe Imager™ (Invitrogen). The concentration and length of the pool were assessed using the Qubit™ 3.0 Fluorometer (Invitrogen) and Bioanalyzer High Sensitivity DNA Kit (Agilent Technologies, Waldbronn, Germany), respectively. The pooled libraries were loaded on an Ion 520™ Chip (Thermo Fisher Scientific, Waltham, MA, USA). Template preparation on Ion Chef and sequencing on the GeneStudio™ S5 System (Thermo Fisher Scientific) were performed, setting the run as described in the Ampli1 LowPass protocol.
5
Germline Mutation Detection using Ion S5
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All the DNA samples were screened for germline mutations using the On-Demand Research Assay on the Ion S5 system (both ThermoFisher Scientific, Waltham, MA, USA). Each DNA sample was checked for concentration using a Qubit 3.0 Fluorometer (Thermo Fisher Scientific). The concentration of input DNA was then adjusted to 50 pmol.
The library and template preparations were performed using the automated Ion Chef System, then sequenced in Ion S5 with Ion 520 Chip (all Thermo Fisher Scientific) according to the manufacturer’s instructions.
Sequencing results were aligned to the hg19 human reference genome and analyzed using the Ion Reporter Software Version 5.10 (Thermo Fisher Scientific).
The variant frequency cut-off for the detection of gene germline mutation was defined as 20%, as recommended by a previous study [7 (link)].
The On-Demand Research Assay showed 99.85% sensitivity, 100% specificity, 0% false-positive rate and 99.99% accuracy in detecting single nucleotide variations (SNVs) and small deletions.
The library and template preparations were performed using the automated Ion Chef System, then sequenced in Ion S5 with Ion 520 Chip (all Thermo Fisher Scientific) according to the manufacturer’s instructions.
Sequencing results were aligned to the hg19 human reference genome and analyzed using the Ion Reporter Software Version 5.10 (Thermo Fisher Scientific).
The variant frequency cut-off for the detection of gene germline mutation was defined as 20%, as recommended by a previous study [7 (link)].
The On-Demand Research Assay showed 99.85% sensitivity, 100% specificity, 0% false-positive rate and 99.99% accuracy in detecting single nucleotide variations (SNVs) and small deletions.
6
BRCA1/2 Genetic Analysis in Breast and Ovarian Cancer
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Peripheral blood samples were harvested at diagnosis from BC or OC patients through a vacutainer syringe containing EDTA. Genomic DNA was isolated using the DNeasy® Blood Kit (QIAGEN, Hilden, Germany). After the extraction phase, DNA has been quantified by Qubit®3.0 fluorometer (Thermofisher Scientific, Waltham, MA, USA) and its quality has been assessed through the use of 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA).
The genetic analysis for BRCA1/2 was performed as previously described (4 (link), 26 (link)).
Sequencing analysis was performed using Ion 520 Chip (Thermofisher Scientific, Waltham, MA, USA) and Ion Torrent S5 (Thermofisher Scientific, Waltham, MA, USA) NGS platform. Obtained data were analyzed using both Amplicon Suite (SmartSeq s.r.l.) and Ion Reporter Software v.5.12 (Thermofisher Scientific, Waltham, MA, USA). NGS data analysis was performed with the standardization of sequencing coverage depth in order to minimize the probability of false positive and negative results in clinical practice, considering a minimum coverage of 500× to each sample.
The genetic analysis for BRCA1/2 was performed as previously described (4 (link), 26 (link)).
Sequencing analysis was performed using Ion 520 Chip (Thermofisher Scientific, Waltham, MA, USA) and Ion Torrent S5 (Thermofisher Scientific, Waltham, MA, USA) NGS platform. Obtained data were analyzed using both Amplicon Suite (SmartSeq s.r.l.) and Ion Reporter Software v.5.12 (Thermofisher Scientific, Waltham, MA, USA). NGS data analysis was performed with the standardization of sequencing coverage depth in order to minimize the probability of false positive and negative results in clinical practice, considering a minimum coverage of 500× to each sample.
7
16S rDNA Amplification for Microbiome Analysis
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For each sample, bacterial 16S rDNA was amplified by polymerase chain reaction (PCR) using the Ion 16S™ Metagenomics Kit (Thermo Fisher Scientific) that uses two primer pools to amplify seven hypervariable regions (V2, V4, V8 and V3, V6-7, V9, respectively). The amplification protocol was as follows: 95 °C for 10 min followed by 25 cycles of 95 °C for 30 s, 58 °C for 30 s and 72 °C for 20 s, and a final step of 7 min at 72 °C. Equal volumes of the two primer reactions were pooled and PCR amplicons purified with paramagnetic beads technology (CleanPCR, Labclinics, Barcelona, Spain). Barcoded libraries were prepared from 5 ng of DNA per sample using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific) to end-repair amplicons and the Ion Xpress™ Barcode Adapters Kit (Thermo Fisher Scientific) to ligate the barcode adapters, according to the manufacturer’s instructions. Libraries were diluted to 22 pM prior to clonal amplification by emulsion PCR with the Ion OneTouch™ 2 System using the Ion 520™ and Ion 530™ Kit-OT2 (Thermo Fisher Scientific), and then sequenced on an Ion S5 System using a Ion 520™ Chip (Thermo Fisher Scientific).
8
Comprehensive IHHNV Genome Sequencing
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We extracted total nucleic acids from tissue homogenates for initial testing by using the MagMAX-Ambion kit 1836 on an automated MagMAX Express magnetic particle processor (ThermoFisher Scientific). We extracted additional DNA from tissue homogenates treated with Benzonase to reduce host DNA by using the DNeasy Blood and Tissue Kit (QIAGEN, https://www.quiagen.com ). We prepared libraries by using the Ion Xpress Plus Fragment Library Kit (ThermoFisher Scientific) according to manufacturer instructions and templates by using the Ion 520 on the Ion Chef System, then sequenced by using an Ion 520 Chip on the Ion S5 System (ThermoFisher Scientific). We performed reference-based mapping (GenBank accession no. NC_002190) by using SeqMan NGen version 14 (DNASTAR, https://www.dnastar.com ) with default parameters for automatic read trimming and assembly and verified alignments resulting in the final consensus sequence. The genomes (minus ends because random priming was used) of IHHNV from Texas and Florida were deposited in GenBank (accession nos. MN968717.1 and MN968716.1).
9
Genetic Analysis of Hereditary Cancer Syndromes
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Peripheral blood was collected from BC, OC, or PC patients. Genomic DNA was extracted from the peripheral blood using the DNeasy® Blood Kit (QIAGEN, Hilden, Germany) and quantified by Qubit®3.0 fluorometer (Thermo Fisher Scientific, Waltham, MA). Its quality was evaluated using 2100 Bioanalyzer (Agilent Technologies, Santa Clara, CA). The genetic analysis for BRCA1/2 was carried out as previously described.2 ,17 ,18 (link) Sequencing analysis was carried out using Ion 520 Chip (Thermo Fisher Scientific) and Ion Torrent S5 (Thermo Fisher Scientific) instrument. The obtained data were processed with two different software called Amplicon Suite (SmartSeq s.r.l., Novara, Italy) and Ion Reporter Software v.5.14 (Thermo Fisher Scientific).
The genetic analysis by multi-gene panel, which included 22 genes involved in risk of hereditary BC, OC, PC, and colorectal cancer, and other inherited cancer syndromes (ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, and TP53), was carried out as previously described.10
The genetic analysis by multi-gene panel, which included 22 genes involved in risk of hereditary BC, OC, PC, and colorectal cancer, and other inherited cancer syndromes (ATM, APC, BARD1, BRCA1, BRCA2, BRIP1, CDH1, CHEK2, EPCAM, MLH1, MSH2, MSH6, MUTYH, NBN, PALB2, PMS2, PTEN, RAD50, RAD51C, RAD51D, STK11, and TP53), was carried out as previously described.10
10
Ion Sequencing of Amplified Honey DNA
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Sequencing of the obtained amplicons was carried out following the protocol already described [41 (link)] with a few modifications. Briefly, PCR products obtained from each honey DNA sample using csd primers were purified with ExoSAP-IT® (USB Corporation, Cleveland, OH, USA) and then sequenced using an Ion S5-Ion Chef System (Thermo Fisher Scientific Inc., Waltham, MA, USA). A total of 12 libraries were produced by end-repair and ligation of the DNA fragments with a specific barcode using the Ion XpressTM Plus Fragment Library and Ion Xpress™ Barcode Adapter 1–32 kits (Thermo Fisher Scientific Inc.). Each library was quantified with the Ion Library TaqMan Quantitation Kit (Thermo Fisher Scientific Inc.) by qPCR with the QuantStudio™ 7 Pro Real-Time PCR System (Thermo Fisher Scientific Inc.). Libraries were first clonally amplified by emulsion PCR and sequenced following the manufacturer’s instructions using the Ion 510™ and Ion 520™ and Ion 530™ Kit-Chef after having pooled them for sequencing in one Ion 520 chip (Thermo Fisher Scientific Inc.).
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