Frozen pellet obtained from 20 ml sample extract after stomaching were centrifuged and DNA extraction was performed on the pellet. Nucleospin® tissue kit (Macherey-Nagel) was used for the extraction according to manufacturer instructions. Concentration and quality of extracted DNA were checked using a BioSpec-nano spectrophotometer (Shimadzu). The bacterial V3-V4 region of the 16S rRNA gene was then amplified using the forward primer 5′-CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG-3′ and the reverse primer 5′-GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT-3′23 (link). The preparation of amplicons was performed in a total volume of 50 µL containing 0.5 µl of TAQ Polymerase (5 U/µl) and 5 µl of adequate 10 X PCR buffer (MTP Taq DNA Polymerase, Sigma), 1 µl of 10 mM dNTP (Sigma), 1.25 µM of each primer (20 µM) and 10 ng of DNA template. PCR was performed using a LightCycler 480 thermocycler as follows: 94 °C for 60 s for initial melting; 30 cycles at 94°c for 60 s, 65 °C for 60 s, 72 °C for 60 s; and 72 °C for 10 min following by incubation at 4 °C. Products were then verified on a 1.5% agarose gel before being sent to GeT-PLaGe Genotoul plateform (Castanet-Tolosan, France) for paired-end sequencing on Illumina MiSeq platform (Illumina, USA) at a read length of 2 × 250 pb.
Mtp taq dna polymerase
Manufactured by Merck Group
Sourced in United States
MTP Taq DNA Polymerase is a thermostable DNA polymerase used for polymerase chain reaction (PCR) amplification of DNA. It is derived from the thermophilic bacterium Thermus aquaticus and possesses 5'-3' polymerase activity and 5'-3' exonuclease activity.
Automatically generated - may contain errors
4 protocols using mtp taq dna polymerase
1
Bacterial 16S rRNA Profiling by Illumina Sequencing
2
16S rRNA Amplification and Sequencing
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16S rDNA was amplified with primers for the V3 and V4 hypervariable regions (PCR1F_460: 5′-CTTTCCCTACACGACGCTCTTCCGATCTACGGRAGGCAGCAG-3′, PCR1R_460:5′-GGAGTTCAGACGTGTGCTCTTCCGATCTTACCAGGGTATCTAATCCT-3′). The reaction mixture contained 10 ng of genomic DNA, 5 U/μl MTP Taq DNA polymerase (Sigma, France), 0.2 mM dNTP, and 0.5 µM (final concentration) of each primer. Reactions were performed using an annealing temperature of 65°C for 30 cycles in a T100 thermocycler (Biorad, France). Sequencing was performed using 460-bp paired-end reads and an Illumina Miseq protocol on the GeT-PLaGe platform (Toulouse, France). Illumina reads were joined using the fastq-join method. The sequences were demultiplexed and quality filtered using the QIIME (version 1.8.0) software package. The sequences were assigned to OTUs using UCLUST algorithm 41 with a 97% threshold of pairwise identity and classified taxonomically using the Greengenes reference database.
3
Microbiome Profiling Using Amplicon Sequencing
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(5'-GCATCGATGAAGAACGCAGC-3') and ITS4
(5'-TCCTCCGCTTWTGWTWTGC-3') primers. The PCR was performed with MTP Taq DNA Polymerase (Sigma-Aldrich, USA), and the cycling conditions were: 94°C for 1 min, followed by 30 cycles of amplification at 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, with a final extension step of 10 min at 72°C. The sequencing was performed with a V3
Illumina MiSeq kit, as described in Poirier et al. (2018) (link) (Poirier et al., 2018) (link).
The quality of the raw data was evaluated with FastQC (Wingett & Andrews, 2018) (link) and the sequences were imported into the FROGS pipeline (Escudié et al., 2018) (link) to obtain the Operational Taxonomic Units (OTUs). The sequences were filtered by length (150-500 bp)
and then pooled into OTUs with SWARM (Mahe, Rognes, Quince, de Vargas, & Dunthorn, 2014) (link) with the distance parameter of 3. Chimeras were removed with VSEARCH (Rognes, Flouri, Nichols, Quince, & Mahe, 2016) (link) and OTUs with at least 0.01% in the whole dataset were retained. The OTUs were affiliated with SILVA 132 SSU databases (Quast et al., 2013) (link) for bacteria and UNITE 8.2 for fungi (https://unite.ut.ee/). Alpha-diversity and beta-diversity analyses were performed in R Studio v.3.6.1 using the phyloseq and ggplot2 packages (v1.30.0) (McMurdie & Holmes, 2013; (link)Poirier et al., 2018) (link).
(5'-TCCTCCGCTTWTGWTWTGC-3') primers. The PCR was performed with MTP Taq DNA Polymerase (Sigma-Aldrich, USA), and the cycling conditions were: 94°C for 1 min, followed by 30 cycles of amplification at 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, with a final extension step of 10 min at 72°C. The sequencing was performed with a V3
Illumina MiSeq kit, as described in Poirier et al. (2018) (link) (Poirier et al., 2018) (link).
The quality of the raw data was evaluated with FastQC (Wingett & Andrews, 2018) (link) and the sequences were imported into the FROGS pipeline (Escudié et al., 2018) (link) to obtain the Operational Taxonomic Units (OTUs). The sequences were filtered by length (150-500 bp)
and then pooled into OTUs with SWARM (Mahe, Rognes, Quince, de Vargas, & Dunthorn, 2014) (link) with the distance parameter of 3. Chimeras were removed with VSEARCH (Rognes, Flouri, Nichols, Quince, & Mahe, 2016) (link) and OTUs with at least 0.01% in the whole dataset were retained. The OTUs were affiliated with SILVA 132 SSU databases (Quast et al., 2013) (link) for bacteria and UNITE 8.2 for fungi (https://unite.ut.ee/). Alpha-diversity and beta-diversity analyses were performed in R Studio v.3.6.1 using the phyloseq and ggplot2 packages (v1.30.0) (McMurdie & Holmes, 2013; (link)Poirier et al., 2018) (link).
4
Microbiome Profiling Using Amplicon Sequencing
Check if the same lab product or an alternative is used in the 5 most similar protocols
+ Expand
(5'-GCATCGATGAAGAACGCAGC-3') and ITS4
(5'-TCCTCCGCTTWTGWTWTGC-3') primers. The PCR was performed with MTP Taq DNA Polymerase (Sigma-Aldrich, USA), and the cycling conditions were: 94°C for 1 min, followed by 30 cycles of amplification at 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, with a final extension step of 10 min at 72°C. The sequencing was performed with a V3
Illumina MiSeq kit, as described in Poirier et al. (2018) (link) (Poirier et al., 2018) (link).
The quality of the raw data was evaluated with FastQC (Wingett & Andrews, 2018) (link) and the sequences were imported into the FROGS pipeline (Escudié et al., 2018) (link) to obtain the Operational Taxonomic Units (OTUs). The sequences were filtered by length (150-500 bp)
and then pooled into OTUs with SWARM (Mahe, Rognes, Quince, de Vargas, & Dunthorn, 2014) (link) with the distance parameter of 3. Chimeras were removed with VSEARCH (Rognes, Flouri, Nichols, Quince, & Mahe, 2016) (link) and OTUs with at least 0.01% in the whole dataset were retained. The OTUs were affiliated with SILVA 132 SSU databases (Quast et al., 2013) (link) for bacteria and UNITE 8.2 for fungi (https://unite.ut.ee/). Alpha-diversity and beta-diversity analyses were performed in R Studio v.3.6.1 using the phyloseq and ggplot2 packages (v1.30.0) (McMurdie & Holmes, 2013; (link)Poirier et al., 2018) (link).
(5'-TCCTCCGCTTWTGWTWTGC-3') primers. The PCR was performed with MTP Taq DNA Polymerase (Sigma-Aldrich, USA), and the cycling conditions were: 94°C for 1 min, followed by 30 cycles of amplification at 94°C for 1 min, 65°C for 1 min, and 72°C for 1 min, with a final extension step of 10 min at 72°C. The sequencing was performed with a V3
Illumina MiSeq kit, as described in Poirier et al. (2018) (link) (Poirier et al., 2018) (link).
The quality of the raw data was evaluated with FastQC (Wingett & Andrews, 2018) (link) and the sequences were imported into the FROGS pipeline (Escudié et al., 2018) (link) to obtain the Operational Taxonomic Units (OTUs). The sequences were filtered by length (150-500 bp)
and then pooled into OTUs with SWARM (Mahe, Rognes, Quince, de Vargas, & Dunthorn, 2014) (link) with the distance parameter of 3. Chimeras were removed with VSEARCH (Rognes, Flouri, Nichols, Quince, & Mahe, 2016) (link) and OTUs with at least 0.01% in the whole dataset were retained. The OTUs were affiliated with SILVA 132 SSU databases (Quast et al., 2013) (link) for bacteria and UNITE 8.2 for fungi (https://unite.ut.ee/). Alpha-diversity and beta-diversity analyses were performed in R Studio v.3.6.1 using the phyloseq and ggplot2 packages (v1.30.0) (McMurdie & Holmes, 2013; (link)Poirier et al., 2018) (link).
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