DNA was extracted from 250-mg fecal samples using the Mo Bio PowerSoil DNA kit (Solana Beach, CA, USA) according to the manufacturer’s instructions. The only modification from the kit protocol was that the bead-beating step was performed twice for 45 s at level 5 on a FastPrep-24 (MP Biomedicals, Solon, OH). The bacterial primers Bakt_341F (CCTACGGGNGGCWGGAG) and Bakt_805R (GACTACHVGGGTATCTAATCC) were used to amplify the bacterial 16S rRNA genes from the isolated DNA. The primers were complemented with 454 adapters and sample-specific barcodes (28 (link)). PCRs were prepared in triplicate and amplified under conditions that have been described elsewhere (29 (link)). PCR products were confirmed using agarose gel electrophoresis, and the triplicate products were pooled and purified using an Agencourt AMPure system (Beckham Coulter Genomics). Purified PCR products were quantified using a Qubit fluorometer (Invitrogen) and mixed into equimolar amounts. The mixed pool of PCR products was sequenced from the reverse primer direction using the Roche/454 GS Titanium technology platform (Branford, CT, USA).
454 gs titanium technology platform
Manufactured by Roche
Sourced in United States
The Roche/454 GS Titanium technology platform is a next-generation DNA sequencing system that utilizes a pyrosequencing approach to generate high-throughput, long-read sequencing data. The core function of this platform is to enable efficient and cost-effective DNA sequencing for a wide range of applications, including de novo genome assembly, targeted resequencing, and metagenomics analysis.
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Lab products found in correlation
2 protocols using 454 gs titanium technology platform
1
16S rRNA Gene Amplicon Sequencing of Fecal Microbiome
2
Fecal Microbiome DNA Extraction and Sequencing
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The fecal samples are subjected to extracting and purifying microbial DNA by a specific commercial DNA kit for purification (QIAamp DNA Stool Mini Kit, Quiagen, Barcelona, Spain). Quantification is conducted with a NanoDrop ND-1000 spectrophotometer (Thermo Fisher Scientific, Newark, DE, USA) in the Department of Microbiology, University Hospital San Cecilio (Granada, Spain). Polymerase chain reaction (PCR) is performed in a FastStart High Fidelity PCR System, dNTP Pack (Roche Applied Science). After PCR, amplicons are further purified using AMPure XP beads (Beckman-Coulter) to remove smaller fragments. DNA concentration and quality are measured using a Quant-iT™ PicoGreen® dsDNA Assay Kit. Afterwards, pyrosequencing of the PCR amplicons is performed using a Roche/454 GS Titanium technology platform (Roche, Branford, CT, USA). The MG-RAST (metagenomics analysis server) and the Ribosomal Database Project are used for the taxonomic analysis. Metagenomics data will be deposited in the publicly available repository MG-RAST (http://metagenomics.anl.gov/ ).
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