Seventeen hereditary cancer genes (ATM, BRCA1, BRCA2, CDKN2A, CHEK2, CTNNB1, ECAD, FANCJ, MLH1, MSH2, MSH6, NBN, PALB2, PTEN, RAD50, RAD51 and TP53) were screened through target capture DNA sequencing using a custom HaloPlex Target Enrichment 1–500 kb design (Agilent, USA) according to the HaloPlex Target Enrichment System-Fast Protocol, Version B. This method was performed only for patient ID–039 for detecting germline mutation in other genes associated with hereditary cancer. The library was sequenced in an Ion PGM Sequencer using an Ion 316 Chip and the Ion PGM Sequencing 200 Kit v2 (Thermo Scientific, Wilmington, DE). The mean targeted base coverage depth was 400X. SNVs and indels were identified using the VariantCaller v4.0.r73742 plugin from the Torrent Suite Browser.
Ion pgm sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion PGM Sequencer is a next-generation DNA sequencing instrument designed for targeted and small-scale genomic analysis. It utilizes semiconductor-based sequencing technology to generate sequencing data. The core function of the Ion PGM Sequencer is to perform DNA sequencing.
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Lab products found in correlation
Ion total rna seq kit v2, by Thermo Fisher Scientific (6 mentions)
Ion pgm sequencing 200 kit v2, by Thermo Fisher Scientific (5 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (5 mentions)
Ion 318 chip, by Thermo Fisher Scientific (5 mentions)
Ion pgm sequencing 400 kit, by Thermo Fisher Scientific (5 mentions)
Ion 318 v2 chip, by Thermo Fisher Scientific (4 mentions)
Ion pgm template ot2 400 kit, by Thermo Fisher Scientific (4 mentions)
Ion pgm template ot2 200 kit, by Thermo Fisher Scientific (3 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (3 mentions)
Ion fragment library kit, by Thermo Fisher Scientific (3 mentions)
Ion onetouch system, by Thermo Fisher Scientific (3 mentions)
Ion pgm hi q sequencing kit, by Thermo Fisher Scientific (3 mentions)
Ion pgm hi q view chef kit, by Thermo Fisher Scientific (2 mentions)
Axyprep mag pcr clean up kit, by Corning (2 mentions)
Ion ampliseq cancer hotspot panel v2, by Thermo Fisher Scientific (2 mentions)
Ion onetouch es system, by Thermo Fisher Scientific (2 mentions)
Ion 316 chip, by Thermo Fisher Scientific (2 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (2 mentions)
Ion pgm 200 sequencing kit, by Thermo Fisher Scientific (2 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (2 mentions)
63 protocols using ion pgm sequencer
1
Hereditary Cancer Genes Profiling
2
Mitogenomic DNA Sequencing via Ion Torrent
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Whole mitogenomic DNA was sequenced using two long DNA fragments with Ion Torrent PGM™ (Thermo Fisher Scientific, MA). The two fragments had adapters with different barcode sequences attached (Ion Xpress Barcode Adapters) that were mixed individually and used as templates for constructing an amplicon library with NEBNext Fast DNA Fragmentation & Library Prep Set (New England Biolabs, MA) for Ion Torrent. The libraries constructed were quantified by using KAPA Library Quantification Kits (KAPA Biosystems, MA) for Ion Torrent and pooled into a single tube. The pooled library sample was further amplified by an emulsion PCR with Ion PGM Template OT2 200 Kit (Thermo Fisher Scientific, MA). The product was finally sequenced on an Ion PGM sequencer (Thermo Fisher Scientific, MA) with Ion 318 Chip Kit v. 2 and Ion PGM Sequencing 200 Kit v. 2.
3
Ion Torrent Sequencing of Multiplexed DNA Libraries
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PCR amplifications from each library were quantified using Qubit™ fluorometer, Quant-IT™ (Invitrogen™, Carlsbad, USA) and prepared to a 1011 DNA molecules/μL. As they included multiplex identifiers, they were equimolecular pooled to continue in one reaction. Ion Torrent adapters were ligated to 30 ng of pooled DNA and libraries were then amplified with Ion Torrent primers for 8 cycles and size selected (2% E-Gel Size Select, Invitrogen). Libraries were sequenced following the manufacturer’s protocol with Ion PGM™ sequencer (Thermo Fisher Scientific, Massachussetts, USA) at DNA sequencing facilities at CRAG (Bellaterra, Spain). Software version for base calling was Torrent-Suite v2.0.1, (Thermo Fisher Scientific, Massachussetts USA). Sequencing data were deposited at European Nucleotide Archive (ENA, http://www.ebi.ac.uk/ena/ ) [26 ] with the accession number PRJEB17083.
4
16S rDNA V4 Amplification and Sequencing
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The 16S rDNA V4 region was amplified by PCR and sequencing as previously described with minor modifications [7 (link)]. FFPE DNA was amplified with the Platinum PCR SuperMix High Fidelity (Thermo Fisher Scientific, Waltham, MA, USA) with forward primer 5′-GTGYCAGCMGCCGCGGTAA-3′ (16S_rRNA_V4_515F) and reverse primer 5′-GGACTACNVGGGTWTCTAAT-3′ (16S_rRNA_V4_806R). PCR products were confirmed by agarose gel electrophoresis and purified with Agencourt AMPure XP reagents (Beckman Coulter, Brea, CA, USA). End repair and barcode adaptors were ligated with an Ion Plus Fragment Library Kit (Thermo Fisher Scientific, Waltham, MA, USA) in compliance with the manufacturer’s instructions, and libraries were constructed. The library concentration was determined with an Ion Library Quantitation Kit (Thermo Fisher Scientific, Waltham, MA, USA), and the same quantity of libraries was set for each sequence. Emulsion PCR and chip loading were performed on the Ion Chef with an Ion PGM Hi-Q View Chef Kit, and sequencing was performed on the Ion PGM Sequencer (Thermo Fisher Scientific, Waltham, MA, USA). The sequence data were transferred to the IonReporter local server with the IonReporterUploader plugin. Data were analyzed with the Metagenomics Research Application using a custom primer set. The analytical parameter was set as the default.
5
Viral Genome Sequencing from Cell Supernatant
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Viral RNA extraction from cell supernatant was performed as described above. A set of specific primer pairs (Table S3 ) was used to produce overlapping amplicons covering the entire viral genome with the SuperScript III One-Step RT-PCR System (Thermo Scientific). Complete genome sequencing was performed using the Ion PGM Sequencer (Thermo Scientific). Read sequences were analyzed as previously described [7 (link)]. To assess the genetic diversity of viral populations, mutation frequency for each position was calculated as the number of reads with a mutation compared to the reference, divided by the total number of reads at that site (minimum coverage of 500). For the analysis, only substitutions with a frequency of at least 1% were taken into account (Table S4 ).
6
16S rRNA Gene Amplification and Sequencing
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Genomic DNA extraction was carried out following the protocol [9 (link)] . The primers 515F and 907R were utilized for PCR amplification of the hypervariable regions V4-V5 of the bacterial 16S rRNA gene. The 907R primer includes a unique barcoded fusion. The primer sequences were: 515F: 5′-GTGCCAGCMGCCGCGGTAA-3′ and 907R: 5′-CCGTCAATTCMTTTRAGT-3′, where M denotes A or C and R denotes purine. The conditions for PCR amplification were: 3 min of denaturation at 94 °C, followed by 25 cycles of 45 s at 94 °C (denaturing), 60 s at 50 °C (annealing), and 90 s at 72 °C (elongation), followed by a final elongation for 10 min at 72 °C. The amplification products were purified by the AxyPrep™ Mag PCR Clean-Up Kit (Axygen, USA). The amplicon libraries were constructed with an Ion Plus Fragment Library Kit (Thermo Fisher Scientific Inc.) [10 ] , then sequenced by the Ion PGM™ Sequencer with the Ion 318™ Chip v2 with a read length of 400 bp (Thermo Fisher Scientific Inc., Ion PGM™ Hi-Q™ OT2 Kit, Cat.No: A27739; Ion PGM™ Hi-Q™ Sequencing Kit, Cat.No: A25592) [11 ] . All experiments were performed in the laboratory of BGI-Shenzhen.
7
Comprehensive Molecular Profiling of FFPE Specimens
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FFPE specimens were received for PCDx testing, and the diagnosis of each case was confirmed on a freshly cut hematoxylin and eosin (HE)-stained slide by a board-certified pathologist (author RJP). Cases were micro/macro dissected when less than 60% tumor cells were present in order to enrich for tumor cells. DNA was extracted from all specimens, and where feasible, ribonucleic acid (RNA) was also extracted. Complementary DNA was created from RNA. A proprietary polymerase chain reaction (PCR)-based method was used to create libraries. All libraries from a given case were simultaneously sequenced on an Ion 318™ chip on the Ion PGM sequencer (Thermo Fisher Scientific, Waltham, MA, USA). mRNA was analyzed for elevated expression at P≤0.001. Copy number variants and alterations were reported. Mutations were compared to a database of mutations, and only those contained within the database were reported. The test was optimized to detect base substitutions with 4% frequency at 99.9% sensitivity and indels with 7% frequency at 99.4% sensitivity. The specificity of mutation assays was optimized to be >99.99% at the patient level, meaning that <0.01% of patient reports will contain a false positive result. A report was generated, reviewed by a board-certified oncologist (author DML) and pathologist (author RJP), signed out, and transmitted to the patient’s physician.
8
Comparative Genomics of Enterococcus faecalis
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Short reads (2,918,486 reads, average length 214 bp) obtained in a previous study using an Ion PGM sequencer (Thermo Fisher Scientific, MA, United States) (Panthee et al., 2021 (link)) were mapped on whole genome sequences of E. faecalis strains listed in Supplementary Table 1 using CLC Genomics Workbench ver. 12.0 (Qiagen, Aarhus C, Denmark) by suffix array algorism (Shrestha et al., 2014 (link)) with the following parameters: length fraction: 0.5 and similarity fraction: 0.8, mapped randomly for non-specific mapping reads. The unmapped reads were collected and assembled de novo by de Bruijn graphs (Gnerre et al., 2011 (link)) with the following parameters: minimum contig length: 250 bp, mismatch cost: 2, insertion cost: 3, deletion cost: 3, length fraction: 0.5, and similarity fraction: 0.8. We selected the contigs longer than 1 kb and confirmed that these contigs were unique to the EF-2001 strain or only a few strains harbored a similar sequence by the Basic Local Alignment Search Tool (BLAST) search of the National Center for Biotechnology Information (NCBI) database. Analysis of phage regions was performed as previously described using the PHAge Search Tool Enhanced Release (PHASTER) (Arndt et al., 2016 (link); Panthee et al., 2021 (link)).
9
Ion AmpliSeq Cancer HotSpot Panel v2 Sequencing
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In this study, an Ion Personal Genome Machine (PGM) Sequencer (Thermo Fisher Scientific) was used as the NGS platform. Library preparation for individual samples was accomplished using the Ion AmpliSeq™ Library Kit 2.0 (Thermo Fisher Scientific) and Ion AmpliSeq™ Cancer HotSpot Panel v2 (Thermo Fisher Scientific), as per the manufacturer’s directions [19 (link)]. The targeted cancer panel sequences 2790 mutations in 50 oncogenes and tumor suppressor genes with known cancer correlations. Generally, 10 ng of genomic DNA from each sample was applied to prepare barcoded libraries, using Ion Xpress™ Barcode Adapters (Thermo Fisher Scientific). Libraries were pooled to a final concentration of 100 pmol/L using the Ion Library Universal Quantification Kit (Thermo Fisher Scientific), and emulsion polymerase chain reaction was performed utilizing the Ion Torrent™ OneTouch™ 2 System. Every pool was loaded onto an Ion 318v2 Chip (Thermo Fisher Scientific) for single-end sequence analysis with an Ion PGM Sequencer, while applying 500 flows (125 cycles) for 200-base-read sequencing.
10
Targeted Gene Panel for Congenital Myopathy
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Patients 1 and 2 were diagnosed by Ion PGM sequencer (Thermo Fisher Scientific, MA) in combination with the original targeted gene panel containing 41 known causative genes for congenital myopathy (Nishikawa, Mitsuhashi, Miyata, & Nishino, 2017 ). We also screened MTM1 pathogenic variant(s) in patient 3 by direct sequencing of the entire coding exons and their flanking regions.
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