One library for each strain (B201, B241, and B250) was prepared using the Ion Total RNA-Seq Kit v2 (Thermo Fisher Scientific) following the manufacturer's instructions with one exception: incubation time for RNA shearing was reduced to 2.5 min. The final cDNA library was quantified with a 2100 Bioanalyzer using a High Sensitivity DNA Chip (Agilent). Library enrichment was carried out with an Ion PGM™ Template OT2 200 kit and Ion OneTouch™ 2 system (Thermo Fisher Scientific). Each library was sequenced with an Ion 318 Chip using an Ion PGM 200 Sequencing kit (Thermo Fisher Scientific) on the Ion Personal Genome Machine (Thermo Fisher Scientific). RNA-Seq data were analyzed by ProteinCT Biotechnologies (Madison, WI, USA). Briefly, raw reads were mapped to the E. coli O157 B201 and B241 genome sequences (Baranzoni et al., 2016 (link)) using the Subjunc program from the Subread-1.4.6 package (http://bioinf.wehi.edu.au/subread ). Raw gene counts were generated by FeatureCounts from the Subread package, and differential expression was calculated using the Limma package (https://bioconductor.org/packages/release/bioc/html/limma.html ). Transcriptomics data are available under NCBI BioProject number PRJNA381969 .
Ion 318 chip
Manufactured by Thermo Fisher Scientific
Sourced in United States
The Ion 318 Chip is a semiconductor-based sequencing chip designed for use with Ion Torrent sequencing systems. It is capable of generating DNA sequence data by detecting the chemical signals released during the sequencing process. The chip provides a platform for high-throughput DNA sequencing, enabling the analysis of genetic samples.
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Lab products found in correlation
Ion pgm 200 sequencing kit, by Thermo Fisher Scientific (18 mentions)
Ion onetouch 2 system, by Thermo Fisher Scientific (11 mentions)
Ion xpress plus fragment library kit, by Thermo Fisher Scientific (8 mentions)
Ion pgm template ot2 200 kit, by Thermo Fisher Scientific (7 mentions)
Ion total rna seq kit v2, by Thermo Fisher Scientific (6 mentions)
Ion torrent pgm, by Thermo Fisher Scientific (6 mentions)
Ion pgm sequencing 200 kit v2, by Thermo Fisher Scientific (6 mentions)
Qiaamp dna mini kit, by Qiagen (5 mentions)
Ion ampliseq library kit 2, by Thermo Fisher Scientific (5 mentions)
Ion pgm sequencer, by Thermo Fisher Scientific (5 mentions)
Ion plus fragment library kit, by Thermo Fisher Scientific (4 mentions)
Ion ampliseq cancer hotspot panel v2, by Thermo Fisher Scientific (4 mentions)
Ion onetouch es instrument, by Thermo Fisher Scientific (4 mentions)
Ion xpress barcode adapter, by Thermo Fisher Scientific (4 mentions)
Ion pgm hi q sequencing kit, by Thermo Fisher Scientific (3 mentions)
Ion onetouch es, by Thermo Fisher Scientific (3 mentions)
Ion onetouch system, by Thermo Fisher Scientific (3 mentions)
Ion ampliseq custom panel, by Thermo Fisher Scientific (3 mentions)
Ion onetouch 200 template kit v2, by Thermo Fisher Scientific (3 mentions)
Ion pgm sequencing 400 kit, by Thermo Fisher Scientific (3 mentions)
61 protocols using ion 318 chip
1
Transcriptome Analysis of E. coli Strains
2
Ion PGM-based Whole-Genome Aneuploidy Analysis
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Templated spheres were loaded on an Ion 318™ chip, and sequencing was performed on an Ion PGM™ running Torrent Suite™ Software 5.0.5 (Thermo Fisher, Waltham, MA, USA) using the Ion PGM Hi-Q Sequencing Kit.
Data were automatically uploaded to Ion Reporter™ Software 5.10 (Thermo Fisher, Waltham, MA, USA) and analyzed using a Low-pass whole-genome aneuploidy workflow, an algorithm that allows copy cumber analysis from a test sample, based on the ratio between the percentage of reads derived from a given chromosome test and a reference value for the same chromosome. As reference value, a custom baseline generated from 11 normal samples was used according to the Ion Reporter™ user manual. A custom baseline was also adopted to reduce the amplification bias and optimize the detection of CNV and consisted of a reference value for each chromosome calculated by averaging the percentage of mapped reads in a series of euploid samples. The CN value with the highest probability for a chromosome was assigned a confidence value of 10. The CNV coverage data were also visualized in the Integrative Genomics Viewer genome browser, launched directly from Ion reporter, which graphically shows the difference in test and control coverages in a ploidy-centric Y-axis. Chromosomal gains were associated with a CN >3 and losses with a copy number <1 [25 (link)].
Data were automatically uploaded to Ion Reporter™ Software 5.10 (Thermo Fisher, Waltham, MA, USA) and analyzed using a Low-pass whole-genome aneuploidy workflow, an algorithm that allows copy cumber analysis from a test sample, based on the ratio between the percentage of reads derived from a given chromosome test and a reference value for the same chromosome. As reference value, a custom baseline generated from 11 normal samples was used according to the Ion Reporter™ user manual. A custom baseline was also adopted to reduce the amplification bias and optimize the detection of CNV and consisted of a reference value for each chromosome calculated by averaging the percentage of mapped reads in a series of euploid samples. The CN value with the highest probability for a chromosome was assigned a confidence value of 10. The CNV coverage data were also visualized in the Integrative Genomics Viewer genome browser, launched directly from Ion reporter, which graphically shows the difference in test and control coverages in a ploidy-centric Y-axis. Chromosomal gains were associated with a CN >3 and losses with a copy number <1 [25 (link)].
3
Sequencing of DNA from Blood Culture
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DNA was extracted from blood culture broth (sample obtained in January 2014) using the manual extraction Gentra Puregene kit (Qiagen CAT# 158389; Autogen INC, Holliston, MA, USA) and quantified using the NanoDrop spectrophotometer. The quality of subjected DNA sample was determined by loading 150 mg of diluted DNA in 1% agarose E-gel (Invitrogen, Paisley, UK). We have conducted 2 sequencing runs using the Personal Genome Machine (PGM) sequencer from Life Technologies (Thermo Fisher, Carlsbad, CA, USA). Briefly, library was prepared using 50 ng of extracted DNA universal primers and AmpliSeq HiFi mix (Thermo Fisher). The 15-cycle amplification product was subjected to digestion using FuPa reagent (Thermo Fisher) and ligated with universal adapters. Purified barcoded libraries were quantified by real-time polymerase chain reaction (qPCR) and normalized to 100 pM. Then, they were subjected to emulsion polymerase chain reaction (ePCR) using the Ion PGM Template OT2 400 and the Ion OneTouch System. The ePCR template was enriched using the OneTouch ES (Thermo Fisher) to produce Ion Sphere particles ready for sequencing as single-ended read library. The library was then loaded to the Ion 318 Chip (Thermo Fisher) and sequenced using the PGM sequencer. The output of the sequencer is a set of single-ended reads with expected maximum length of 400 bp.
4
Microbiome Sequencing Using Ion Torrent PGM
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The Ion Torrent PGM (Thermo Fisher Scientific, Waltham, USA) protocol for end repair, barcode and adapter attachment, clonal amplification, and sequencing was applied for the amplified fragments V1V2, V4V5, and V6V7V8 of the 16S rRNA gene (0–2.000 GE of E. coli, S. enterica, and V. cholerae) (Table S3). Samples containing human DNA were processed by the addition of 30 ng of previously fragmented human DNA of approximately 350 bp in length. The DNA was fragmented using the Bioruptor standard sonicator (Diagenode, Denville, USA) following the protocol recommended by the manufacturer. Fragments of 350 bases were selected using E-Gel SizeSelect on the E-Gel iBase Power System (Thermo Fisher Scientific, Waltham, USA). The libraries were constructed using the Ion Xpress Plus Fragment Library Kit (Life Technologies, Carlsbad, USA) followed by emulsion PCR on the Ion OneTouch 2 System using the Ion PGM Hi-Q Template Kit (Thermo Fisher Scientific, Waltham, USA). Enrichment was performed on the Ion OneTouch ES (Thermo Fisher Scientific, Waltham, USA), and sequencing was conducted on the Ion 318 Chip.
5
Ion Torrent PGM Sequencing Protocol
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Emulsion PCR and sequencing were performed according to the manufacturer's instructions. The detailed protocol was described elsewhere.9 (link) MPS was performed with an Ion Torrent Personal Genome Machine (PGM) system using an Ion PGM 200 Sequencing Kit and an Ion 318 Chip (Thermo Fisher Scientific).
6
Custom Ion AmpliSeq Panel for Targeted Sequencing
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Sequencing panel was custom designed with the Ion AmpliSeq ™ Designer online tool (www.ampliseq.com ) targeting exons and UTR from the 10 selected genes (padding: ± 5bp). This design allowed the analysis of 165 amplicons varying from 125 to 375 base pairs.
DNA samples were isolated from peripheral blood using Gentra Puregene Kit (Qiagen‐ Sciencesm). Libraries were constructed using Ion AmpliseqTM Library Kit 2.0 and normalized using Ion Library EqualyzerTM Kit according to the manufacturer's protocol. Amplified libraries were then submitted to emulsion polymerase chain reaction (PCR) on the Ion OneTouchTM 2 system using the Ion PGMTM Hi‐QTM OT2 Kit (Thermo Fisher) following manufacturer's protocol. Ion sphere particles were enriched on the Ion OneTouch ES (Thermo Fisher), loaded on an Ion 318 chip (Thermo Fisher) and sequenced on the Ion Torrent PGMTM (Thermo Fisher).
Exons and UTR from the 10 selected candidate genes were sequenced in the cohort of 73 patients in two separate runs.
DNA samples were isolated from peripheral blood using Gentra Puregene Kit (Qiagen‐ Sciencesm). Libraries were constructed using Ion AmpliseqTM Library Kit 2.0 and normalized using Ion Library EqualyzerTM Kit according to the manufacturer's protocol. Amplified libraries were then submitted to emulsion polymerase chain reaction (PCR) on the Ion OneTouchTM 2 system using the Ion PGMTM Hi‐QTM OT2 Kit (Thermo Fisher) following manufacturer's protocol. Ion sphere particles were enriched on the Ion OneTouch ES (Thermo Fisher), loaded on an Ion 318 chip (Thermo Fisher) and sequenced on the Ion Torrent PGMTM (Thermo Fisher).
Exons and UTR from the 10 selected candidate genes were sequenced in the cohort of 73 patients in two separate runs.
7
Targeted Amplicon Sequencing for Genomic Profiling
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Genomic DNA was isolated using QIAamp DNA Mini Kit (Qiagen, Hilden, Germany) according to manufacturer's instructions. A DNA sample from each subject was used for sequencing by Ampliseq ultra-high multiplex PCR (IonAmpliseq Library kit; Life Technologies, Carlsbad, CA). Custom primer sets were designed using the Ion Ampliseq Designer tool (Life Technologies). The respective genes sequenced in this study are summarized in Table 2 . The library after purification, equalization, and the template prep was processed for the Ion 318 Chip on the Ion OneTouch system (Thermo Fisher Scientific, Waltham, MA). Final sequencing was conducted using the Ion PGM Next-Generation Sequencing Platform (Thermo Fisher Scientific, Waltham, MA).
Reads from Ampliseq amplicon sequencing were aligned to the reference human genome (NCBI37/hg19) using the Bowtie2 alignment tool [19 (link)]. Single nucleotide variants were called at each amplicon using SAMtools and BCFtools [20 (link)]. Possible genotypes and their likelihoods were computed at each position with SAMtools mpileup, from which variants and genotypes were called using BCFtools [19 (link)].
Reads from Ampliseq amplicon sequencing were aligned to the reference human genome (NCBI37/hg19) using the Bowtie2 alignment tool [19 (link)]. Single nucleotide variants were called at each amplicon using SAMtools and BCFtools [20 (link)]. Possible genotypes and their likelihoods were computed at each position with SAMtools mpileup, from which variants and genotypes were called using BCFtools [19 (link)].
8
Hearing Loss Genes Targeted Sequencing
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Amplicon libraries of the target exons were prepared with an Ion AmpliSeq™ Custom Panel (ThermoFisher Scientific, MA, USA) designed using Ion AmpliSeq™ Designer (https://www.ampliseq.com ) for 63 or 68 genes (listed in Table S1 ) (Kitano et al., 2017 ; Nishio et al., 2015 ) reported to cause nonsyndromic hearing loss (Hereditary Hearing loss Homepage; http://hereditaryhearingloss.org/ ) with the Ion AmpliSeq™ Library Kit 2.0 (ThermoFisher Scientific) and Ion Xpress™ Barcode Adapter 1‐96 Kit (ThermoFisher Scientific) according to the manufacturer's instructions. After the amplicon libraries were prepared, equal amounts of the libraries for six patients were pooled for one Ion PGM™ sequence reaction and those for 45 patients were pooled for one Ion Proton™ sequencing.
The emulsion PCR was performed with the Ion OneTouch™ System and Ion OneTouch 200 Template Kit v2 (ThermoFisher Scientific) or Ion PI™ Hi‐Q™ OT2 200 Kit according to the manufacturer's instructions. Sequencing was performed with an Ion torrent PGM™ system using the Ion PGM™ 200 Sequencing Kit and Ion 318™ Chip (ThermoFisher Scientific), or Ion Proton™ system using the Ion PI™ Hi‐Q™ Sequencing 200 Kit and Ion PI™ Chip (ThermoFisher Scientific) according to the manufacturer's instructions.
The emulsion PCR was performed with the Ion OneTouch™ System and Ion OneTouch 200 Template Kit v2 (ThermoFisher Scientific) or Ion PI™ Hi‐Q™ OT2 200 Kit according to the manufacturer's instructions. Sequencing was performed with an Ion torrent PGM™ system using the Ion PGM™ 200 Sequencing Kit and Ion 318™ Chip (ThermoFisher Scientific), or Ion Proton™ system using the Ion PI™ Hi‐Q™ Sequencing 200 Kit and Ion PI™ Chip (ThermoFisher Scientific) according to the manufacturer's instructions.
9
Ion PGM Library Preparation and Sequencing
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Before sequencing, the template preparation for library pools was performed either with Ion OneTouch 2 system (Thermo Fisher Scientific) using the Ion PGM™ Hi-Q™ OT2 Kit (Thermo Fisher Scientific) or Ion Chef system (Thermo Fisher Scientific) using the Ion PGM™ Hi-Q™ Chef Kit following the kit protocols. 2 µl of each 10 pM library was used for library pool, and 15–20 libraries were pooled together. 20 µl of a library pool was mixed with 5 µl of nuclease-free water and added to the amplification solution for template preparation. After the template preparation, the quality of resulting ion spheres was checked with Qubit 3.0 fluorometer (Thermo Fisher Scientific). Following quality check, the ion spheres were loaded on an Ion 318™ Chip (Thermo Fisher Scientific) and sequenced with Ion PGM system using Ion PGM Hi-Q Sequencing kit (Thermo Fisher Scientific) according to the protocol provided with the kit.
10
Gut Microbiota Analysis in Probiotic-Treated Children
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To analyze the gut microbiota, paired stool samples were collected at onset of INS and during treatment with probiotics from eight probiotic-treated children. In addition, stool samples were also obtained from 21 healthy children (median age 4.0 years). Stool samples were stored at −80 °C until analysis. The frozen stool specimens were thawed and prepared for 16S rRNA sequencing using the DNA Stool Kit (Macherey-Nagel, Düren, Germany). The seven hypervariable regions of 16S rRNA, except v1 and v5, were sequenced, and the 16S Metagenome Kit (Thermo Fisher Scientific, Waltham, MA, USA) was used for amplification. After purification, the libraries were constructed using the Ion Plus Fragment Library Kit (Thermo Fisher Scientific) and the Ion Xpress Barcode Adapters Kit (Thermo Fisher Scientific). Barcoded libraries were quantitated using an Agilent Bioanalyzer 2000 (Agilent, Santa Clara, CA, USA) and pooled at a final concentration of 30 pM per target. Emulsion polymerase chain reaction and target enrichment for template preparation were performed using the Ion Chef Instrument Kit (Thermo Fisher Scientific). Sequencing was performed using an Ion PGM sequencer (Thermo Fisher Scientific) and an Ion 318 chip (Thermo Fisher Scientific). All resulting sequence data were analyzed using Ion Reporter software (Thermo Fisher Scientific).
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