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Molecular Diagnostic Solutions in Algal Cultivation Systems
Published in Stephen P. Slocombe, John R. Benemann, Microalgal Production, 2017
Laura T. Carney, Robert C. McBride, Val H. Smith, Todd W. Lane
There are several molecular markers that have been employed for molecular analysis of clinical and environmental samples (Hoef-Emden 2012) including the large and small subunit ribosomal RNA (LSU and SSU rRNA, respectively) genes, the internal transcribed spacer (ITS) of the ribosomal RNA genes, and the mitochondrial cytochrome oxidase (cox) gene. The best known of these targets is the SSU rRNA gene (see Figure 8.1). The prokaryotic SSU rRNA gene has nine hypervariable regions dispersed along its length (Van De Peer et al. 1996). Of these, the sequence information contained in the individual hypervariable regions 3 or 6 (Huse et al. 2008) or a fragment covering hypervariable regions 1 through 3 or 4 (Kim et al. 2011) are the most useful for phylogenetic determination. Sequence data from individual hypervariable regions are often sufficient for genus-level distinction, whereas data from multiple hypervariable regions or the entire SSU rRNA gene can result in species-level distinction.
Metabarcoding approach to identify bacterial community profiling related to nosocomial infection and bacterial trafficking-routes in hospital environments
Published in Journal of Toxicology and Environmental Health, Part A, 2023
Bárbara Gimenes de Castro, Bruno Mari Fredi, Rafael dos Santos Bezerra, Queren Apuque Alcantara, Carlos Eduardo Milani Neme, Daniele Enriquetto Mascarelli, Aline Seiko Carvalho Tahyra, Douglas dos-Santos, Camilla Rizzo Nappi, Fernanda Santos de Oliveira, Flavia Pereira Freire, Giulia Ballestero, Julia Beatriz Menuci Lima, Juliana de Andrade Bolsoni, Juliana Lourenço Gebenlian, Naira Lopes Bibo, Nathália Soares Silva, Nilton de Carvalho Santos, Victoria Simionatto Zucherato, Kamila Chagas Peronni, Daniel Guariz Pinheiro, Emmanuel Dias-Neto, Gilberto Gambero Gaspar, Valdes Roberto Bollela, Vanessa da Silva Silveira, Aparecida Maria Fontes, Nilce Maria Martinez-Rossi, Svetoslav Nanev Slavov, João Paulo Bianchi Ximenez, Fernando Barbosa, Wilson Araújo Silva
Two different and independent samples were collected from the environment (Table 1 and Figure 1). The sample was organized according to the MetaSUB Sampling Tutorial recommendations (MetaSUB International Consortium 2016). This biological material was acquired with a 0.9% saline solution swab. All duplicate samples from the same location were then mixed. DNA was extracted using the DNeasy PowerSoil Kit protocol (Qiagen C) following the manufacturer’s instructions. DNA concentration and purity were quantified in a NanoDrop ND-1000 spectrophotometer (NanoDrop Technologies, Rockland, DE). At the same time, DNA integrity was examined using the Agilent 2100 Bioanalyzer. The V3-V4 hypervariable region of the 16S rRNA gene was amplified and prepared for sequencing. The libraries were sequenced in a MiSeq system using a V2kit with a single-end 300 nt run. The library preparation followed the 16S Metagenomic Sequencing Library protocol (Preparing 16S Ribosomal RNA Gene Amplicons for the Illumina MiSeq System) defined for the Illumina Miseq sequencing platform. The library was then followed by sequencing using the Illumina MiSeq system and bioinformatics data analysis.
Metagenome based analysis of groundwater from arsenic contaminated sites of West Bengal revealed community diversity and their metabolic potential
Published in Journal of Environmental Science and Health, Part A, 2023
Anumeha Saha, Abhishek Gupta, Pinaki Sar
Total DNA was extracted from 2 L of groundwater using Epicentre Metagenomic DNA isolation kit, following manufacturer protocol post filtration of groundwater through 0.1 µm membrane filter. DNA concentration was determined using NanoDrop 1000 spectrophotometer and Qubit 3.0 fluorometer (Invitrogen, Thermo Fisher Scientific). Post quantification, total DNA samples were subjected to amplification of the V4 hypervariable region of 16S rRNA gene using barcoded V4 specific primer set 515 F/806R.[33] PCR reaction was performed in 25 µL volume using Amplitaq Gold 360 Mastermix (Applied Biosystems), 40 pmol of barcoded forward and reverse primers each, 10-50 ng of metagenomic DNA under the following program: Initial denaturation 95 °C for 10 min followed by 35 cycles of 95 °C for 35 sec, 50 °C for 40 sec, 72 °C for 40 sec and a final extension at 72 °C for 10 min. PCR amplified products were run on 2% SizeSelect E-gel (Invitrogen). Amplicon library was prepared, further processed, and subjected to sequencing on in-house Ion Torrent Next-Generation (Ion S5) Sequencing platform using Ion 530™ Chip Kit and Ion 520™ and Ion 530™ Kit-OT2 (Thermo Fisher Scientific).
Microbial consortia adaptation to substrate changes in anaerobic digestion
Published in Preparative Biochemistry & Biotechnology, 2022
Priyanka S. Dargode, Pooja P. More, Suhas S. Gore, Bhupal R. Asodekar, Manju B. Sharma, Arvind M. Lali
The purified DNA samples were outsourced to Xcelris Labs Ltd., India for 16S rRNA amplicon sequencing. The 16S rRNA bacterial and archaeal libraries were prepared using Nextera XT Index Kit (Illumina Inc., USA) as per the 16S Metagenomic Sequencing Library Preparation protocol (Part # 15044223 Rev. B). Primers for the amplification of the V3-V4 hypervariable region (Table 4) of the 16S rRNA gene of bacteria and archaea were designed and synthesized in the Xcelris facility. The amplicon libraries were purified by 1X AMpureXP beads and checked on Agilent DNA 1000 chip on Bioanalyzer 2100 and quantified on a fluorometer by Qubit dsDNA HS Assay kit (Life Technologies, USA). The libraries were then sequenced using the Illumina sequencing chemistry to generate ∼150 Mb of data per one 16S bacterial library and ∼100 Mb of data per one archaeal library. QIIME (Quantitative Insight into Microbial Ecology) tool was used for analyzing 16S metagenome data from NGS platforms.