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454 gs junior titanium platform

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The 454 GS Junior Titanium platform is a next-generation sequencing (NGS) system designed for targeted sequencing applications. It utilizes 454 sequencing technology, which is a type of pyrosequencing method. The 454 GS Junior Titanium platform is capable of generating high-quality, long sequencing reads, making it suitable for various genomic research applications.

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Lab products found in correlation

Agencourt ampure xp pcr purification system, by Beckman Coulter (1 mentions) Quant it picogreen dsdna kit, by Thermo Fisher Scientific (1 mentions) Ultraclean fecal dna bead tubes, by Qiagen (1 mentions) Dneasy blood and tissue kit, by Qiagen (1 mentions)

Close spelling variants of this product

454 GS Junior Titanium 454 GS Junior platform 454 GS Junior 454-Junior platform GS Junior platform 454 Titanium platform GS Junior Roche 454 platform Titanium platform

4 protocols using 454 gs junior titanium platform

1

Sequencing Six Ontario Virus Genomes

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The complete nucleotide sequences of 6 viruses were determined using the 454 GS Junior Titanium platform (Roche Applied Science, Indianapolis, IN, USA) as described by Grgic et al. [21 (link)]. Briefly, RNA of all 6 Ontario 2012 viruses was fragmented by ZnCl2 into fragments between 500 bp and 1500 bp. Sheared RNA was used for first and second strand cDNA synthesis and for fragment end repair. Double-stranded cDNA purification was performed using AMPure beads and Magnetic Particle Concentrator (MPC). Once the End Repair program was completed the 454 rapid library multiplex identifier (RL MID) was ligated to the fragments according to the GS Junior Titanium cDNA rapid library preparation method (Roche). Quality assessment of the RNA samples was performed using the FlashGel system. Sequence assembly was done with the Newbler (version 2.5p1) de novo sequence assembly software (Roche).
Grgić H., Costa M., Friendship R.M., Carman S., Nagy É, & Poljak Z. (2015). Genetic Characterization of H1N1 and H1N2 Influenza A Viruses Circulating in Ontario Pigs in 2012. PLoS ONE, 10(6), e0127840.
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2

Rapid Virus Genome Sequencing with 454 GS Junior

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The complete nucleotide sequences of 10 viruses were determined using the 454 GS Junior Titanium platform (Roche Applied Science, Indianapolis, IN, USA). In brief, RNA of all viruses was fragmented by ZnCl2 into fragments between 500 bp and 1500 bp. Sheared RNA was used for first and second strand cDNA synthesis and fragment end repair. The 454 rapid library multiplex identifier (RL MID) was ligated to the fragments according to the GS Junior Titanium cDNA rapid library preparation method (Roche) by incubating with ligase at 25C° for 10 min. Quality assessment of the RNA samples and of the DNA library was performed using the FlashGel system. The nucleotide sequence reads obtained were assembled using the Newbler (version 2.5p1) de novo sequence assembly software (Roche). Comparison of the de novo contigs by BLAST with known influenza A virus sequences allowed identification of all genes. Sanger sequencing applying targeted oligo primers was utilized to walk across gaps. BLASTN and BLASTX analyses were performed to compare the established sequences to known influenza A viruses in the NCBI database.
Grgić H., Costa M., Friendship R.M., Carman S., Nagy É., Wideman G., Weese S, & Poljak Z. (2014). Molecular characterization of H3N2 influenza A viruses isolated from Ontario swine in 2011 and 2012. Virology Journal, 11, 194.
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3

Colonic Microbiome Profiling via 16S rRNA Sequencing

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The DNeasy blood and tissue kit (Qiagen) was used to extract genomic DNA from colonic tissue samples. The extraction was performed according to the manufacturer’s instructions except for the following modifications: adding a bead-beating step using UltraClean fecal DNA bead tubes (Mo Bio Laboratories), doubling the amount of ATL buffer and the proteinase K used in the protocol, and decreasing by half the amount of the AE buffer used to elute the DNA. Subsequently, the V3, V4, and V5 hyper-variable regions of the 16S rRNA gene in each of the samples were targeted for amplification with the 357F and 929R primer sets.59 (link) Amplicons were purified with the Agencourt AMPure XP PCR purification system (Beckman Coulter), and quantified with the Quant-iT PicoGreen dsDNA kit (Life Technologies) to obtain an equal pool for pyrosequencing. They were then sequenced on a Roche 454 GS Junior Titanium platform according to the manufacturer’s specifications. Bacterial 16S sequences were first processed using the microbial ecology software suite mothur60 (link) to generate operational taxonomic units (OTUs) at a 3%, i.e., species level of difference. These data, in the form of the .shared file, were then imported into the R software and analyzed using the R-package vegan. The inverse Simpson diversity measure was calculated using the function diversity().
McDermott A.J., Frank C.R., Falkowski N.R., McDonald R.A., Young V.B, & Huffnagle G.B. (2014). Role of GM-CSF in the inflammatory cytokine network that regulates neutrophil influx into the colonic mucosa during Clostridium difficile infection in mice. Gut microbes, 5(4), 476-484.
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16S rRNA Gene Amplicon Sequencing

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Gene amplicons representing the V3–V5 hypervariable region of the bacterial 16S rRNA encoding gene were generated and analyzed as previously described (Hashway et al., 2014 (link)) per protocols developed for the NIH Human Microbiome Project (http://www.hmpdacc.org/doc/16S_Sequencing_SOP_4.2.2.pdf). Pyrosequencing was performed on the Roche 454 GS Junior Titanium platform according to the manufacturer’s instructions (Roche 454 Life Sciences, Branford, CT, USA).
Wilding L.A., Bassis C.M., Walacavage K., Hashway S., Leroueil P.R., Morishita M., Maynard A.D., Philbert M.A, & Bergin I.L. (2015). Repeated dose (28 day) administration of silver nanoparticles of varied size and coating does not significantly alter the indigenous murine gut microbiome. Nanotoxicology, 10(5), 513-520.
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