The concentration and purity of the metagenomic DNA extracted were measured using a spectrophotometer (NanoDrop 2000, Thermo Scientific, Waltham, MA, USA). Approximately, 400 bp DNA fragments of the bacterial 16S rRNA gene targeting the hypervariable region V3–V4 were amplified using the primer pair 341F (5′‐CCTACGGGNGGCWGCAG‐3′) and 805R (5′‐GACTACHVGGGTATCTAATCC‐3′) fused with the Illumina MiSeq adaptors and a 6 bp barcode sequence unique to each sample (Tian et al., 2015 ). The PCR amplification products were subsequently purified, combined in equimolar ratios, and subjected to high‐throughput sequencing on an Illumina MiSeq sequencing platform to produce paired 250‐nucleotide reads at Sangon Biotech (Shanghai, China).
Miseq adaptors
Manufactured by Illumina
Sourced in United States
MiSeq adaptors are laboratory equipment designed to be used with the MiSeq Sequencing System. They are used to prepare DNA samples for sequencing on the MiSeq platform. The adaptors facilitate the attachment of DNA fragments to the flow cell, enabling the sequencing process.
Automatically generated - may contain errors
Lab products found in correlation
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
C1000 touch thermocycler, by Bio-Rad (2 mentions)
Miseq sequencing platform, by Illumina (1 mentions)
Accuprime high fidelity pcr kit, by Thermo Fisher Scientific (1 mentions)
Nextera xt library preparation, by Illumina (1 mentions)
Ampure bead, by Beckman Coulter (1 mentions)
Phusion 2 mastermix, by Illumina (1 mentions)
4 protocols using miseq adaptors
1
Metagenomic 16S rRNA Sequencing Protocol
2
16S rRNA Gene Profiling of Biofilm Communities
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To characterise biofilm communities, 16S rRNA gene V4 sequences were PCR-amplified from 1 μl of DNA extract using the AccuPrime High Fidelity PCR kit (Invitrogen Catalog No 12346094) with the primer pair 515F (5′ AGCMGCCGCGGTAA 3′) and 806R (5′ GGACTACHVGGGTWTCTAAT ′3) containing Illumina MiSeq adaptors and single-end barcodes. PCR temperature cycles weres: 98 °C for 3 s, 33 cycles of: 98 °C for 20 s, 50 °C for 30 s, 72 °C for 90 s; then 72 °C for a final 10 min. Amplicons were pooled in equal quantities, cleaned with AMPure beads (Beckman Coulter) and paired-end sequenced on the MiSeq platform following Nextera XT library preparation (Illumina).
3
16S rRNA Gene V4 Amplification
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For each sample, the V4 hypervariable region of the 16S rRNA gene was amplified using previously validated primers that contained dual-end adapters for indexing as previously described (22, 23) . Briefly, PCR reactions were set-up and performed in triplicate using the 515F forward and 806R reverse rRNA gene V4 primers (24) with Illumina MiSeq adaptors. A Bio-Rad C1000 Touch Thermocycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) was used for PCR amplification with the following parameters: 94°C for 3 min followed by 30 cycles of 94°C for 45 sec, 50°C for 60 sec, and 72°C for 90 sec, with a final incubation at 72°C for 10 min.
4
16S rRNA Gene Amplification: Detailed Protocol
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For each sample, the amplification of the V4 hypervariable region of the 16S rRNA gene was conducted using previously validated primers containing dual-end adapters for indexing (Nelson et al., 2014 (link); Benjamino et al., 2018 (link)). The PCR reactions were prepared with a total volume of 83.4 μl, consisting of 3 μl of BSA (New England Biosciences (NEB), Ipswich, MA, USA), 41.7 μl of Phusion 2× Master Mix (NEB), 2.5 μl of a 10 μM primer mix (515F forward and 806R reverse rRNA gene V4 primers with Illumina MiSeq adaptors), and 30 ng of sample DNA. To ensure accuracy, each sample underwent triplicate assays using a Bio-Rad C1000 Touch Thermocycler (Bio-Rad Laboratories Inc., Hercules, CA, USA) with the following cycling parameters: an initial denaturation at 94°C for 3 min, followed by 30 cycles of 45 s at 94°C, 60 s at 50°C, and 90 s at 72°C. A final elongation step of 10 min at 72°C concluded the process.
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