Onetouch es

Manufactured by Thermo Fisher Scientific

Sourced in United States

The OneTouch ES is a laboratory equipment product designed for sample preparation and analysis. It provides a standardized and automated solution for various laboratory workflows. The core function of the OneTouch ES is to assist in the efficient handling and processing of samples, ensuring consistent and reliable results.

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6 protocols using onetouch es

Ion Torrent Sequencing Protocol

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Barcoded samples were pooled in equimolar ratios to a total concentration of 9 pM in low TE buffer. Template preparation and enrichment was performed using an Ion OneTouch Template 200 Kit (Life Technologies, NY, USA) on a OneTouch 2 and OneTouch ES (Life Technologies, NY, USA). Sequencing was performed using an Ion PGM 200 Sequencing Kit on “316” sequencing chips for a total of 500 nucleotide flows, yielding average read lengths of 220–230 bp. Five or six samples were pooled on a single chip, generally yielding >450,000 reads per sample.

Warrilow D., Watterson D., Hall R.A., Davis S.S., Weir R., Kurucz N., Whelan P., Allcock R., Hall-Mendelin S., O’Brien C.A, & Hobson-Peters J. (2014). A New Species of Mesonivirus from the Northern Territory, Australia. PLoS ONE, 9(3), e91103.

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Ion Torrent 16S Amplicon Sequencing

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16S amplicons from all samples and controls were pooled into one of four sequencing libraries in equimolar amounts. Amplicon libraries were then purified twice using 1.2 volumes of Agencourt Ampure XP beads (Agilent Technologies, USA) and quantified by qPCR using a known concentration of a serially diluted 152 bp synthetic oligonucleotide as a standard. qPCR reactions contained 1X Power Syber Green mastermix (Life Technologies, USA), 0.4 μM Ion Torrent primers A and P1, and 2 μl DNA template, and were run with the following thermal conditions: initial denaturation at 95 °C for 5 min followed by 30 cycles of denaturation at 95 °C (30 s), annealing and extension at 60 °C (45 s). Templating emulsion PCR and enrichment were performed according to the manufacturer’s recommendations on the One-Touch 2 and One-Touch ES instruments (Life Technologies, USA). Sequencing was performed on an Ion Torrent PGM (Life Technologies, USA) using 400 bp chemistry and 316-V2 semiconductor chips, following the manufacturer’s recommendations.

Gofton A.W., Oskam C.L., Lo N., Beninati T., Wei H., McCarl V., Murray D.C., Paparini A., Greay T.L., Holmes A.J., Bunce M., Ryan U, & Irwin P. (2015). Inhibition of the endosymbiont “Candidatus Midichloria mitochondrii” during 16S rRNA gene profiling reveals potential pathogens in Ixodes ticks from Australia. Parasites & Vectors, 8, 345.

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Ion Torrent RNA-Seq Library Preparation

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The quality of RNA samples was evaluated using a Bioanalyzer 2100 (Agilent). ERCC Spike-In Mix2 was added to each RNA sample prior to poly-A mRNA extraction using a Dynabeads mRNA Purification Kit (Ambion). RNA-seq library preparations were carried out using an Ion Total RNA-Seq Kit v2 (Life Technologies) according with the manufacturer’s instructions with modifications. Chemical 5-min-long RNA fragmentation was used instead of an enzymatic treatment to increase the reproducibility and proportion of long fragments. Size selection using Caliper LabChip XT (Perkin-Elmer) was carried out to obtain library inserts 250–300 bp long. E-PCR, enrichment and quantification for Ion Torrent sequencing were performed with One-Touch 2 and One-Touch ES systems (Life Technologies). Sequencing was carried out on the Ion PGM (Life Technologies) using Hi-Q View sequencing kits and 318v2 chips. ERCC analysis demonstrated the absence of significant misrepresentation (R-squared values, 0.93–0.97).

Kochetov A.V., Glagoleva A.Y., Strygina K.V., Khlestkina E.K., Gerasimova S.V., Ibragimova S.M., Shatskaya N.V., Vasilyev G.V., Afonnikov D.A., Shmakov N.A., Antonova O.Y., Gavrilenko T.A., Alpatyeva N.V., Khiutti A, & Afanasenko O.S. (2017). Differential expression of NBS-LRR-encoding genes in the root transcriptomes of two Solanum phureja genotypes with contrasting resistance to Globodera rostochiensis. BMC Plant Biology, 17(Suppl 2), 251.

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Small RNA Sequencing from Plasma

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RNA was isolated from plasma samples using protocol described earlier30 (link). SmallRNA libraries generated using the Ion Total RNA-Seq Kit v2 were processed through emulsion PCR using OneTouch 2 (Life Technologies) and the Ion PGM™ Template OT2-200 Kit, then enriched using the OneTouch ES (all Life Technologies). One sample was loaded per Ion-318^™ chip and sequenced on Ion PGM using the Ion PGM™ Sequencing 200 Kit v2 (all Life Technologies). The aligned BAM files were analyzed using Strand NGS (Strand Life Sciences) and differential miRNA expression was determined by a two-fold change in normalized read number.

Farr R.J., Januszewski A.S., Joglekar M.V., Liang H., McAulley A.K., Hewitt A.W., Thomas H.E., Loudovaris T., Kay T.W., Jenkins A, & Hardikar A.A. (2015). A comparative analysis of high-throughput platforms for validation of a circulating microRNA signature in diabetic retinopathy. Scientific Reports, 5, 10375.

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Targeted Exome Sequencing of BRCA2 VUS

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Genomic DNA (40 ng) was amplified using 250 primer pairs (GeneRead DNASeq Targeted panels v2, Qiagen) to target all of the exonic regions as well as the intronic regions within 20-bp of a splicing junction. Amplicons were ligated to a barcode adaptor using the Ion Xpress™ Plus Fragment Library Kit (Life Technologies, Carlsbad, CA, USA). The barcoded library was then enriched by emulsion PCR using OneTouch2 and OneTouch ES instruments (Life Technologies) following the Ion Torrent protocol provided by Life Technologies. The library's quantity and quality were examined on a fragment analyzer (Advanced Analytical Technologies) and a Qubit fluorometer (Invitrogen). The enriched library was sequenced using the Ion Personal Genome Machine (PGM) with an Ion 318 chip (Life Technologies) following the manufacturer's instructions. The mean sequencing depth for FFPE tumor samples was >5700×, with a mean uniformity of 91.1%. Variants with a frequency >10% were confirmed by Sanger sequencing of tumor DNA. Germline DNA was analyzed by NGS or Sanger sequencing. Germline DNA from the siblings of the two sisters carrying the BRCA2 VUS p.S1946P was also examined by Sanger sequencing.

Chao A., Chang T.C., Lapke N., Jung S.M., Chi P., Chen C.H., Yang L.Y., Lin C.T., Huang H.J., Chou H.H., Liou J.D., Chen S.J., Wang T.H, & Lai C.H. (2016). Prevalence and clinical significance of BRCA1/2 germline and somatic mutations in Taiwanese patients with ovarian cancer. Oncotarget, 7(51), 85529-85541.

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Single-Cell RNA-Seq of Coronavirus Samples

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Individual corona cell samples were isolated for RNAsequencing using the PicoPure® RNA Isolation Kit (Life Technologies, Carlsbad CA) with modifications. Briefly, samples were lysed and bound to a silica-based filter, treated with RNase-free DNase I (Qiagen, Valencia CA) and washed several times before being recovered in 20 μL elution solution. Purified cDNA libraries were constructed from 50 ng total RNA, which was rRNA-depleted using the Ion Total RNA-Seq Kit v2, Whole Transcriptome Library Prep protocol (Life Technologies, Carlsbad CA). rRNA-depleted total RNA was fragmented using RNase III and purified by Agencourt Ampure XP micro-beads (Beckman Coulter Inc., Brea CA). The fragmented RNA was then hybridized and ligated before reverse transcription and addition of multiplexing barcode adaptor. The subsequent cDNA was then amplified by polymerase chain reaction and purified again. Barcoded libraries were equalized and pooled into an evenly represented final library consisting of 100 pM cDNA, which was then coupled onto templated capture beads and enriched using the Ion OneTouch 2 and OneTouch ES systems (Life Technologies, Carlsbad CA). Final libraries were prepped for loading on the ION PI v2 chip and then sequenced on the Ion Proton with a P1 200 v2 Sequencing kit (Life Technologies, Carlsbad CA).

Parks J.C., Patton A.L., McCallie B.R., Griffin D.K., Schoolcraft W.B, & Katz-Jaffe M.G. (2016). Corona cell RNA sequencing from individual oocytes revealed transcripts and pathways linked to euploid oocyte competence and live birth. Reproductive biomedicine online, 32(5).

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