To examine bacterial community composition within all samples, the 16S rRNA gene was amplified with the universal forward primer 27F (5′-AGAGTTTGATCMTGGCTCAG-3′) and the universal reverse primer 519R (5′-CGGTTACCTTGTTACGACTT-3′) (Weisburg et al., 1991 (link)). PCR reactions were performed in 25 μL volumes containing 12.5 μL GoTaq Green Master Mix, 0.4 μL of each primer (10 μM), and 2 μL of template DNA. PCR cycling conditions involved an initial activation step at 95°C for 120 s, followed by 30 cycles of: denaturation at 95°C for 30 s, annealing at 50°C for 30 s and extension at 72°C for 90 s, followed by a holding stage at 72°C for 10 min. The resultant amplicons were visualized on 1% agarose gel with GelRed (1:10000). Genomic DNA was used to prepare DNA libraries with the Illumina TruSeq DNA library preparation protocol. Sequencing was performed on the Illumina MiSeq platform (at Molecular Research LP, Shallowater, TX, United States) following the manufacturer’s guidelines. Subsequently generated raw data files were deposited in the Sequence Read Archive (SRA) under BioProject number PRJNA342246 (Hurtado-McCormick, 2018a ).
Truseq dna library preparation protocol
Manufactured by Illumina
Sourced in United States
The TruSeq DNA library preparation protocol is a laboratory workflow designed to prepare DNA samples for sequencing. It provides a standardized method for constructing DNA libraries from various sample types. The protocol includes steps for DNA fragmentation, end repair, adapter ligation, and PCR amplification to generate the final sequencing-ready library.
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Lab products found in correlation
Hotstartaq plus master mix kit, by Qiagen (25 mentions)
Miseq platform, by Illumina (8 mentions)
Ampure xp bead, by Beckman Coulter (7 mentions)
Powersoil dna isolation kit, by Qiagen (3 mentions)
Ampure xp bead, by Illumina (2 mentions)
Nanodrop, by Thermo Fisher Scientific (2 mentions)
Rnalater, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000, by Thermo Fisher Scientific (2 mentions)
Nanodrop 2000 2000c spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Miseq next generation sequencer, by Illumina (1 mentions)
High throughput sequencing, by Illumina (1 mentions)
Qiaamp dna stool mini kit, by Qiagen (1 mentions)
Nucleospin tissue kit, by Macherey-Nagel (1 mentions)
Mr dna, by Illumina (1 mentions)
Hotstartaq plus master mix, by Qiagen (1 mentions)
Nanodrop nd 2000 uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions)
Fastdna spin kit for soil, by MP Biomedicals (1 mentions)
Miseq sequencing platform, by Illumina (1 mentions)
Wizard genomic dna purification, by Promega (1 mentions)
Miseq instrument, by Illumina (1 mentions)
36 protocols using truseq dna library preparation protocol
1
16S rRNA Gene Amplification for Bacterial Community Analysis
2
Microbial Community Profiling by 16S rRNA Gene Sequencing
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Microbial genomic DNA was extracted using PowerSoil® DNA isolation kit (MoBio, Carslbad, CA, USA) as per Earth Microbiome Project protocols (Marotz et al., 2017 (link)). Community composition was evaluated as per protocols and primers described (McHugh and Schwartz, 2016 (link)) that targeted archaeal and bacterial hypervariable V4 region (515f/806r) of 16S rRNA gene (Caporaso et al., 2011 (link)). Amplicons were generated using HotStarTaq Plus Master Mix Kit (Qiagen) by following subsequent conditions for amplification: initial denaturation (94 °C for 3 min) followed by 30 cycles, each at 94 °C for 30 s, 53 °C for 40 s, and 72 °C for 1 min, with a final elongation step at 72 °C for 5 mins. PCR products were analyzed on 2% agarose gel. Multiple samples were pooled in equal proportions based on DNA concentration and molecular weight. The pooled samples were purified by calibrated Ampure XP beads and used to prepare DNA libraries following Illumina TruSeq DNA library preparation protocol. Sequencing was performed at the Molecular Research DNA laboratory (Shallowater, TX, USA) on a MiSeq (Illumina) platform in an overlapping 2 × 300 bp configuration with a minimum throughput of 20,000 reads for each sample.
3
Bacterial 16S rRNA Gene Amplicon Sequencing
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DNA extractions of all samples were done according to the instructions in the PowerSoil DNA Isolation Kit from MoBio (Carlsbad, CA, USA). Samples were measured by Nanodrop (ThermoFisher Scientific, Carlsbad, CA, USA). Successful amplification of the bacterial 16S rRNA gene (primers 8F-1492R) was checked on a 1% agarose gel, alongside an extraction blank. Samples were successful and the extraction blank failed as expected. Full amplicon sequencing was performed at the Molecular Research DNA lab (Shallowater, TX, USA) using bacterial primers 27F-519R, with the barcode on the forward primer. A total of 30 cycles of PCR were performed using HotStarTaq Plus Master Mix Kit (Qiagen, Valencia, CA, USA) under the following conditions: 94 °C for 3 min, followed by 28 cycles of 94 °C for 30 s, 53 °C for 40 s and 72 °C for 1 min, and a final elongation step at 72 °C for 5 min. Samples were pooled in equal proportions and purified using calibrated Ampure XP beads. Then the purified PCR product was prepared for sequencing via Illumina TruSeq DNA library preparation protocol and sequenced on the MiSeq platform (Illumina, San Diego, CA, USA). Sequence reads were joined via the Molecular Research pipeline.
4
Microbial Community Analysis of Sediment Samples
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The DNA of sediment samples was extracted using E.Z.N.A Soil DNA kit (OMEGA, United States) according to the manufacturer protocol and stored at −20°C until further analysis. DNA extracts were checked for quantity and purity with NanoDrop™ 2000/2000c Spectrophotometers (Thermo Fisher Scientific) and subsequently submitted to the sequencing platform Mr. DNA (Molecular Research LP, Shallowater, TX). The 16S ribosomal RNA (rRNA) gene V4 variable region PCR primers 515/806 for Eubacteria and Archaea with barcode on the forward primer were used in a 30 cycle PCR using the HotStarTaq Plus Master Mix Kit (Qiagen, United States) under the following conditions: 94°C for 3 min, followed by 28 cycles of 94°C for 30 s, 53°C for 40 s, and 72°C for 1 min, after which a final elongation step at 72°C for 5 min was performed. After amplification, PCR products were checked in 2% agarose gel to determine the success of amplification and the relative intensity of bands. PCR products were used to prepare a DNA library following Illumina TruSeq DNA library preparation protocol. Sequencing was performed on Illumina MiSeq Next Generation Sequencer in accordance with the manufacturer guidelines.
5
16S rRNA Microbiome Analysis of Faecal and Mucosal Samples
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Genomic DNA (gDNA) was extracted from faecal and mucosal samples by using QIAamp® DNA Stool Mini Kit (Qiagen, Courtabœuf, France) and NucleoSpin® Tissue Kit (Macherey-Nagel, Hoerdt, France), respectively, following the manufacturer’s instructions. Five mice per group were chosen for 16S rRNA gene analysis by high-throughput sequencing (Illumina) based on both the histological score and the DNA quantity and quality. The V4 variable region of the 16S rRNA genes was amplified by PCR using universal primers F515 (5′-GTGCCAGCMGCCGCGGTAA-3′) and R806 (5′-GGACTACHVGGGTWTCTAAT-3′)54 (link). The DNA library was constructed following TruSeq DNA library preparation protocol (Illumina, San Diego, CA, USA). Paired-end sequencing (2 × 250 bp) was performed at MR DNA (www.mrdnalab.com , Shallowater, TX, USA) on a MiSeq platform.
6
Genomic Fragment Library Preparation
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Genomic fragment libraries were prepared at the Australian Genome Research Facility with the Illumina TruSeq DNA library preparation protocol (40 (link)). Random subsets of 1 million read pairs were selected to perform read mapping and de novo assembly for comparative analysis against the published M4 GAS strain MGAS10750 (RefSeq accession number NC_008024) (32 (link)).
7
Fungal Community Profiling via Illumina Sequencing
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Diversity assay using bTEFAP Illumina MiSeq (2 × 300 PE) was performed at Molecular Research LP (MR DNA, www.mrdnalab.com , Shallowater, TX, USA) using the primer set ITS-5 and ITS-4 (White et al., 1990 ) and DNA extracted at the previous section as template. Barcode (CACACTCA) was added on 3′ of the forward primer. PCR product was checked in a 2 % (w/v) agarose gel where the presence of the band, as well as their relative intensity, was determined. The sample was purified using calibrated Ampure XP beads. Then, the purified PCR products were used to prepare the DNA library by following Illumina TruSeq DNA library preparation protocol. Sequencing was performed following the manufacturer's guidelines. The sequence data derived from the sequencing process was processed using MR DNA analysis pipeline (MR DNA, Shallowater, TX, USA).
8
Microbial Community Analysis via 16S rRNA Sequencing
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The total DNA (fish + pathogen DNA) extracted from the field samples was amplified using a bacteria-specific primer set with 27F (5′-GAGTTTGATCMTGGCTCAG-3′) and 519R (5′-GWATTACCGCGGCKGCTG-3′), flanking the hypervariable regions V1–V3 of the 16S rRNA gene [59 (link)] with a barcode on the forward primer. Amplification was carried out using the Qiagen Kit HotStarTaq Plus Master Mix under the conditions described in Mandakovic et al. (2018) [60 (link)]. After amplification, the PCR products were visualized in a 2% agarose gel to determine the success of the amplification and the relative intensity of the bands. At this point, the three barcoded samples for the microbial analyses were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled samples were purified using calibrated Agencourt AMPure XP beads and used to prepare the DNA libraries following the Illumina TruSeq DNA library preparation protocol. The sequencing was performed at the molecular research DNA laboratory (Shallowater, TX, USA) on an Illumina MiSeq platform in an overlapping 2 × 300 bp configuration with a minimum throughput of 20,000 reads per sample.
9
Soil Microbiome DNA Extraction and Sequencing
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DNA was extracted from 250 mg of each rhizosphere soil sample using the FastDNA Spin Kit for Soil (MP Biomedicals, Fountain Parkway, Solon, OH, USA), according to the manufacturer’s instructions. DNA concentration and purity were determined using a Nanodrop ND-2000 UV-VIS spectrophotometer (NanoDrop Technologies, Wilmington) and DNA samples were stored at -20 °C.
Amplification and sequencing of bacterial and archaeal DNA were performed by MR DNA (www.mrdnalab.com , Shallowater, TX, USA) by targeting the V4 hypervariable region of the 16 S rRNA gene. Bacterial DNA was amplified by using the universal primers 515 F/806R [52 (link)], while archaeal DNA was amplified with the primers 349 F/806R [53 (link)]. After amplification, the quality and relative concentration of the amplicons were checked by migration on 2% agarose gel. Multiple replicates were pooled together in equal proportions based on their molecular weight and DNA concentrations. Pooled DNA samples were purified using calibrated Ampure XP beads and then the amplicons were used to prepare DNA libraries following Illumina Truseq DNA library preparation protocol. Sequencing was performed on a MiSeq Illumina platform (2 × 300) following the manufacturer’s guidelines.
Amplification and sequencing of bacterial and archaeal DNA were performed by MR DNA (
10
16S rRNA Gene Amplification and Sequencing
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An approximately 300-bp fragment from the V4 variable region of the 16S rRNA genes was amplified from the microbial mock DNA mixture and the soil genomic DNA by PCR using “universal” primers F515 (GTGCCAGCMGCCGCGGTAA) and R806 (GGACTACHVGGGTWTCTAAT) [16 (link)]. Two sequencing libraries were then constructed following the TruSeq DNA library preparation protocol (Illumina), and sequencing of the amplicons was performed on the MiSeq platform (2 × 300 bp, Illumina).
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