DNA was extracted from bacteria and EVs in stools using a stool DNA extraction kit (Bioneer, Daejeon, Korea). Bacterial genomic DNA was amplified with 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 518R (5′-ATTACCGCGGCTGCTGG-3′) primers, which are specific for V1-V3 hypervariable regions of 16S rDNA gene. The libraries were prepared using PCR products according to the GS FLX titanium library prep guide and quantified using a Picogreen assay (Invitrogen, Waltham, USA). After PCR products extracted and quantified, equimolar ratios from each mixture were pooled and sequenced on a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer’s recommendations.
454 life sciences genome sequencer flx system
Manufactured by Roche
Sourced in Switzerland
The 454 Life Sciences Genome Sequencer FLX system is a high-throughput DNA sequencing platform. It utilizes pyrosequencing technology to determine the nucleotide sequence of DNA samples. The system is capable of generating large amounts of sequencing data in a parallel manner.
Automatically generated - may contain errors
Lab products found in correlation
Picogreen assay, by Thermo Fisher Scientific (1 mentions)
Stool dna extraction kit, by Bioneer (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Precellys 24 homogenizer, by Bertin Technologies (1 mentions)
Qiaquick gel extraction kit, by Qiagen (1 mentions)
Quantifluor st fluorometer, by Promega (1 mentions)
Agilent 2100 bioanalyzer, by Agilent Technologies (1 mentions)
Nanodrop nd 1000 spectrophotometer, by Promega (1 mentions)
4 protocols using 454 life sciences genome sequencer flx system
1
16S rDNA Profiling of Gut Microbiota
2
16S rRNA Gene Amplification from Fecal Samples
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Frozen faecal samples were thawed, and bacterial genomic DNA was extracted from 200 mg of feces using QIAamp® DNA Stool Mini Kit (Qiagen, Hilden, Germany) according to the manufacturer's instructions, with the additional glass-bead beating steps on a Precellys 24 homogenizer (Bertin Technologies, Montigny, France). The bacterial genomic DNA was amplified in 50-μl triplicates with the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) primers specific for the V1-V3 hypervariable regions of the 16S rRNA gene according to our previous studies54 (link). Each forward primer incorporated FLX Titanium adapters and a sample barcode at the 5′ end of the reverse primer to allow all samples to be included in the single 454 FLX sequencing run. After PCR products extracted and quantified, equimolar concentrations were pooled and sequenced on a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer's recommendations.
3
16S rRNA Gene Amplification and Sequencing
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The bacterial genomic DNA was amplified with the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) primers specific for the V1-V3 hypervariable regions of the 16S rRNA gene33 (link)34 (link)45 (link). Each forward primer incorporated FLX Titanium adapters and a sample barcode at the 5′ end of the reverse primer to allow all samples to be included in the single 454 FLX sequencing run. All PCR reactions were performed in 50-μl triplicates and combined after PCR. The products were extracted with the QIAquick Gel Extraction Kit (QIAGEN) and quantified on NanoDrop ND-1000 spectrophotometer, QuantiFluor-ST Fluorometer (Promega, USA) and Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA). Equimolar concentrations of 83 samples were pooled and sequenced on a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer’s recommendations.
4
16S rRNA Gene Amplification and Sequencing
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The bacterial genomic DNA was utilized in 50-μl triplicate samples for the specific amplification of the V1-V3 hypervariable regions of the 16S rRNA gene using the 27F (5′-AGAGTTTGATCCTGGCTCAG-3′) and 533R (5′-TTACCGCGGCTGCTGGCAC-3′) primers, as described in our previous reports35 (link)36 (link). Each forward primer incorporated FLX Titanium adapters and a sample barcode was incorporated at the 5′ end of the reverse primer to allow all of the samples to be included in the single 454 FLX sequencing run. After the PCR products were extracted and quantified, they were pooled in equimolar concentrations and were sequenced using a 454 Life Sciences Genome Sequencer FLX system (Roche, Basel, Switzerland) according to the manufacturer’s recommendations. The methods for sequence screening, diversity analysis and taxonomy-based analysis were performed as described in our previous reports35 (link)36 (link). The Mann–Whitney U test and a one-way ANOVA were used for statistical analysis, and all of these analyses were performed using SPSS for Windows software (version 16.0; SPSS Inc., Chicago, IL, USA).
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