Composition and diversity of nrfA genes in sediment samples were examined with a barcode pyrosequencing method using the Ion Torrent PGM sequencer. PCR was conducted in duplicate with each 20 μL sample reaction containing the nrfA2Faw and nrfAR1 primers modified to include 8-bp barcode (forward primers) (Hamady et al., 2008 (link)) and adapter sequences for the Ion Torrent PGM sequencer (Life Technologies, Grand Island, NY). The primer sequences used for pyrosequencing are listed in Supplementary Table 1 . PCR reactions were carried out using Platinum PCR Supermix (Life Technologies, Grand Island, NY) with the following PCR cycle: an initial 5 min at 94°C, 40 cycles of 94°C for 30 s, 52°C for 45 s, and 72°C for 20 s, followed by 5 min at 72°C. PCR results, generating 290 base pair fragments, were checked by running an aliquot on a 2% agarose gel. Duplicate reactions were combined, and amplicons were purified using UltraClean GelSpin DNA Purification Kit (Mo-Bio, Carlsbad, CA). The concentration of purified PCR products was measured using a 2200 TapeStation instrument and D1K reagents (Agilent Technologies, Santa Clara, CA) following the manufacturer's instruction. Pyrosequencing was conducted using an Ion Torrent PGM sequencer with barcode samples pooled on 316 chips, following the Ion Torrent 400 bp sequencing kit protocol (Life Technologies, Grand Island, NY).
Ion torrent pgm sequencer
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion Torrent PGM sequencer is a benchtop DNA sequencing instrument that utilizes semiconductor technology to detect the incorporation of nucleotides during the sequencing process. It provides rapid and cost-effective DNA sequencing for a variety of applications.
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Lab products found in correlation
Ion sphere quality control kit, by Thermo Fisher Scientific (4 mentions)
Qubit 3.0 fluorometer, by Thermo Fisher Scientific (3 mentions)
2100 bioanalyzer, by Agilent Technologies (3 mentions)
Ion torrent onetouch system, by Thermo Fisher Scientific (2 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (2 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (2 mentions)
Ion pgm hi q view sequencing kit, by Thermo Fisher Scientific (2 mentions)
Ion 316 chip, by Thermo Fisher Scientific (2 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (2 mentions)
Ion xpress barcode adapter, by Thermo Fisher Scientific (2 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (2 mentions)
Ion xpresstm plus fragment library kit, by Thermo Fisher Scientific (2 mentions)
E gel size select agarose gel, by Thermo Fisher Scientific (2 mentions)
Ion xpress plus fragment library kit reagent kit, by Thermo Fisher Scientific (2 mentions)
Experion automated electrophoresis system and experion dna 1 k reagents and supplies, by Bio-Rad (2 mentions)
Experion dna chips kits, by Bio-Rad (2 mentions)
Ion pgm hi q view ot2 kit reagents, by Thermo Fisher Scientific (2 mentions)
Dynabeads myone streptavidin c1 magnetic particles, by Thermo Fisher Scientific (2 mentions)
Agencourt ampure xp magnetic particles, by Beckman Coulter (2 mentions)
Qiaamp dna mini kit, by Qiagen (2 mentions)
40 protocols using ion torrent pgm sequencer
1
Examining nrfA Gene Diversity in Sediments
2
Targeted Gene Sequencing Workflow
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Example 7
A library was prepared using an AmliSeq system (Thermo Fisher Scientific) for regions comprising exons of the VAV1 gene or prepared using an Ion Plus Fragment Library kit (Thermo Fisher Scientific) for PCR amplicons amplified by genomic PCR using KOD Plus neo (TOYOBO). After sequencing was conducted with an Ion Torrent PGM sequencer (Thermo Fisher Scientific) in accordance with a standard protocol for 300 base pairs, mutation candidates were analyzed by Variant caller software and their results were confirmed by direct sequencing. The results obtained are shown in
3
Transcriptome Assembly and Annotation from Molluscan Tissues
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We prepared 100-bp DNA libraries from the mRNA samples (1–5 μg each sample) that were extracted from the mantle and the dart sac tissues using an Ion Total RNA-seq Kit v2 (Thermo Fisher Scientific) according to the manufacturer’s protocols, and analyzed them using an Ion 318 v2 chip of the Ion Torrent PGM sequencer (Thermo Fisher Scientific), then performed 100-base single-end sequencing. We obtained a total of 6,056,290 and 5,351,015 raw reads from the mantle and the dart sac tissues, respectively (supplementary table S1 , Supplementary Material online). We then combined these reads and assembled them using Newbler v2.8 (Roche, Basel, Switzerland) under default conditions for cDNA assembly (runAssembly -o output -cdna -large sff-file), and obtained a total of 74,293 contigs. Quality of the assembled sequences was calculated with the BUSCO v2 (Simao et al. 2015 (link)) (supplementary table S1 , Supplementary Material online). We then filtered the contigs to collect contigs longer than 100 bp (59,618 contigs), and used them for our analyses. These shot-gun sequences (DRA006965 and DRA006966) and assembled sequences (PRJDB6927: IADG01000001–IADG01059618) are available in the DNA Data Bank of Japan (DDBJ).
4
Ion PGM Sequencing Protocol
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Based on the qPCR quantification of libraries, equimolar amounts of each library were pulled for a final combined molarity of 400 pM. The emPCR was run by using the Ion PGM Template OT2 200 Kit (Thermo Fisher Scientific) and loaded in the Ion OneTouch™ 2 instrument. Next, non-templated Ion Sphere Particles (ISP) beads were eliminated by magnetic bead purification. The mixture of barcoded libraries was sequenced with the Ion PGM Sequencing 200 Kit V2 (Thermo Fisher Scientific). The sequencing beads were loaded in Ion 318 v2 or 316 v2 Chips (Thermo Fisher Scientific), and sequenced on the Ion Torrent™ PGM sequencer (Thermo Fisher Scientific).
5
Validating Cancer Variant Detection Using Hotspot Control
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The Acrometrix Oncology Hotspot Control DNA (Thermo Fisher, Waltham, MA; Cat. No. 969056) was used for generating sequencing data for testing and evaluating MGE and ELECTRO as previously described [22 (link)]. This control DNA contains more than 500 COSMIC mutations from 53 genes (Thermo Fisher, Waltham, MA; Cat. No. 969056). A targeted cancer panel with selected 25 genes (NextDay Seq-Pan Cancer HotSpot Panel kit, CureSeq Inc.) was used for a deep sequencing of the The Acrometrix Oncology Hotspot Control DNA as previously described [22 (link),34 (link)]. In brief, 10 ng of control DNA was amplified and ligated with barcoded and adaptors with the library prep kit (NextDay Seq-Pan Cancer HotSpot Panel kit, CureSeq Inc.). The prepared library was cleaned up using magnetic beads and resuspended in 1x low-TE buffer, followed by qualitative and quantitative electrophoretic analysis by High Sensitivity DNA chip (Agilent Technologies, Santa Clara, CA; Cat. No. 5067–4626). After the emulsion PCR and library enrichment, the prepared library was sequenced using an Ion 314 v2 Chip (Thermo Fisher), and sequenced on an Ion Torrent™ PGM sequencer. The generated sequencing data was processed with MGE from data submission to data distribution as described above.
6
Bacterial Genomic DNA Extraction and Sequencing
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The genomic DNA was extracted using the GenElute™ Bacterial Genomic DNA Kit (Sigma, USA) following the manufacturer instructions. The concentration and purity of genomic DNA was measured using dsDNA HS (High Sensitivity) Assay Kit in Qubit 3.0 fluorometer (Thermo Fisher Scientific Inc., Waltham, MA, USA) and subsequently DNA quality was visualized by agarose gel electrophoresis.
Genomic libraries were prepared by using the NEB Next Fast DNA Fragmentation and Library Preparation Kit, developed for Ion Torrent (New England Biolabs) and used according to 200 bp protocol. After chemical fragmentation, DNA size selection was performed on precast 2% E-Gel Size Select Gel (Thermo Fisher Scientific Inc., Waltham, MA, USA). The quality of the libraries was verified using Agilent high sensitivity DNA assay kit (Agilent Technologies Inc., Santa Clara, CA, USA) in Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., Santa Clara, CA, USA). For the template preparation, Ion PGM Hi-Q View OT2 Kit was used (Thermo Fisher Scientific Inc., Waltham, MA, USA). The template positive beads were loaded on Ion 316v2 Chip and sequenced using Ion PGM Hi-Q View Sequencing Kit on Ion Torrent PGM sequencer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
Genomic libraries were prepared by using the NEB Next Fast DNA Fragmentation and Library Preparation Kit, developed for Ion Torrent (New England Biolabs) and used according to 200 bp protocol. After chemical fragmentation, DNA size selection was performed on precast 2% E-Gel Size Select Gel (Thermo Fisher Scientific Inc., Waltham, MA, USA). The quality of the libraries was verified using Agilent high sensitivity DNA assay kit (Agilent Technologies Inc., Santa Clara, CA, USA) in Agilent 2100 Bioanalyzer System (Agilent Technologies Inc., Santa Clara, CA, USA). For the template preparation, Ion PGM Hi-Q View OT2 Kit was used (Thermo Fisher Scientific Inc., Waltham, MA, USA). The template positive beads were loaded on Ion 316v2 Chip and sequenced using Ion PGM Hi-Q View Sequencing Kit on Ion Torrent PGM sequencer (Thermo Fisher Scientific Inc., Waltham, MA, USA).
7
Genomic Library Preparation and Sequencing of S. sonnei
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Isolation and genomic library preparation of S. sonnei 4351 was performed as described previously [34 (link)], employing the Qiagen DNeasy Plant Mini Kit (Qiagen, Germantown, MD, USA), the Ion Xpress Plus Fragment Library Kit, and the Ion 316 Chip with the Ion Torrent PGM sequencer (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the recommendation of the manufacturer. Whole transcriptome sequencing was also performed using Ion Torrent Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific Inc., Waltham, MA, USA) according to the manufacturer’s recommendation.
8
Targeted NGS of Hematological Malignancies
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Genomic DNA was extracted from bone marrow or peripheral blood samples at the onset of disease diagnosis by using Invitrogen DNA Extraction Kit. The mutational hotspots or whole coding regions of 51 genes (Supplementary Table 1
9
16S rRNA Gene Amplicon Sequencing
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For taxonomic identification, ~2 × 103 sorted cells from the main bacterial populations were added to 30 μl of Q5 High Fidelity Master Mix (New England BioLabs) complemented with primers and nuclease-free water (Ambion). The V3-V4 hyper-variable regions (490 bp) of 16S rRNA gene were PCR-amplified using S-D-Bact-0341-b-S-17 and S-D-Bact-0785-a-A-21 primers30 (link) tailed with the Ion Torrent sequencing adapters. The forward primer also included the PGM barcode adapter (Ion Xpres Barcode Adapters 1–96 Kit, Thermo- Fisher Scientific). PCR products (~490 bp) were gel purified with NucleoSpin Gel and PCR Cleanup kit (Macherey-Nagel, Düren), pooled and used as template for emulsion PCR with the Ion Torrent One-Touch System (ThermoFisher Scientific) at a concentration of 26 pmol l−1. Sequencing of PCR products was done on an Ion Torrent PGM sequencer (ThermoFisher Scientific) using the Hi-Q sequencing chemistry. Sequences were quality trimmed for sequence length (>300 bp), controlled for homopolymers (<2%) and ambiguities (<2%) and separated by barcode using mothur31 (link). Taxonomic affiliation was extracted by sequence comparison to the SSU rRNA SILVA database 119 using the SILVAngs pipeline32 (link).
10
Next-Generation Sequencing Library Preparation
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Obtained PCR products were used to prepare libraries for diversity analyses by next generation sequencing (NGS) approach on Personal Genome Machine (Life Technologies, Carlsbad, CA, USA) according to Milani et al. [71 (link)]. 200 ng of DNA from each sample was used to prepare sequencing libraries by NEBNext® Fast DNA Library Prep Set kit (New England Biolabs, Ipswich, MA, USA) according to manufacturer’s protocol. The Ion Xpress Barcode adapters (Thermo Fisher Scientific, Waltham, MA, USA) were used to label each sample. The adaptor ligated libraries were purified and simultaneously size-selected using AMPure XP bead sizing (Beckman Coulter, Brea, CA, USA). The barcoded libraries were pooled in equimolar amount (about 26 pM). The pool of libraries was used to prepare sequencing template by emulsion PCR on Ion Sphere Particles (ISPs) using Ion PGMTM Hi-QTM View OT2 400 Kit (Thermo Fisher Scientific) in Ion OneTouchTM 2 instrument. The enrichment of the template positive ISPs were performed on Ion OneTouchTM ES instrument. The enriched template positive ISPs were then loaded in Ion 316TM Chip v2 BC (Thermo Fisher Scientific). The sequencing was then performed on an Ion Torrent PGM sequencer (Thermo Fisher Scientific, Waltham, MA, USA) using Ion PGMTM Hi-QTM View Sequencing solutions kit (Thermo Fisher Scientific).
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