PCR amplification of the 16S rRNA gene (V5eV3 regions) was performed using universal barcoded primers (357F and 926R) and the AccuPrime Taq DNA polymerase (Invitrogen, Waltham, MA, USA) in triplicate. The PCR protocol included an initial denaturation step (95°C for 2 mins) followed by 30 cycles of 95°C for 20 seconds, 50°C for 30 seconds, and 72°C for 5 minutes as described in our prior study [11 (link)]. Individual amplicon libraries were purified using Agencourt AMPure XP (Beckman Coulter, Inc., Indianapolis, IN, USA) beads, quantified using the Quant-iT PicoGreen dsDNA quantitation kit (Invitrogen), and normalized for pooling. Amplicon libraries were sequenced using the 454 GS Junior Titanium (Roche, Indianapolis, IN, USA).
454 gs junior titanium
Manufactured by Roche
Sourced in United States, Switzerland
The 454 GS Junior Titanium is a compact next-generation sequencing system designed for targeted, small-scale sequencing applications. It utilizes Roche's 454 sequencing technology to generate high-quality, long sequencing reads in a rapid and efficient manner. The system is intended for use in research laboratories and small-scale genomic projects.
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Lab products found in correlation
Quant it picogreen dsdna quantitation kit, by Thermo Fisher Scientific (1 mentions)
Accuprime taq dna polymerase, by Thermo Fisher Scientific (1 mentions)
Agencourt ampure xp, by Beckman Coulter (1 mentions)
Abi 3500 genetic analyzer, by Thermo Fisher Scientific (1 mentions)
454 gs junior platform, by Roche (1 mentions)
2 protocols using 454 gs junior titanium
1
16S rRNA Gene Amplification and Sequencing
2
Targeted NGS for Leukemia Mutation Profiling
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NGS, including data analysis was performed for 30 leukemia-associated target genes on the 454 GS Junior platform with 454 GS Junior Titanium chemistry for amplicon sequencing (Roche Diagnostics, Basel, Switzerland) as previously described. 13, 14 In total, 231 amplicons were prepared for each sample and processed in a single NGS run. Mutations with a frequency >15% were confirmed using Sanger Sequencing with standard techniques on an ABI 3500 Genetic Analyzer (Thermo Fisher Scientific, Waltham, MA, USA) as described previously. 14 DNA-sequencing raw data is available on reasonable request.
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