DOI: 10.6084/m9.figshare.11930892
title: DATA FROM: Discovery of Thermophilic Bacillales using Reduced-Representation Genotyping for Identification.

description: <b>Abstract</b>This data set contains complexity-reduced genotyping short-read sequences (length 30-69 bp) of 99 bacterial isolates obtained from samples collected between 2015 and 2016 from domestic hot water systems in the Australian Capital Territory (ACT), commercial composts available in the ACT and artesian water bores located in the Birdsville track in South Australia. These bacterial isolates were identified to belong to the genera of <i>Anoxybacillus</i>, <i>Geobacillus </i>or <i>Brevibacillus</i>. The data is shown as filtered fastA files. The research used two pairs of restriction enzymes <i>Pst</i>I with <i>Hpa</i>II and <i>Pst</i>I with <i>Mse</i>I.<br><b>Keywords</b>bacterial identification; DArTseq; genotyping-by-sequencing; Great Artesian Basin; reduced-representation sequencing; thermophiles<br><b>Specifications table:</b><b>Subject area: </b>Environmental Microbiology<b>Type of data:</b> Short-read sequences<b>How data was acquired: </b>Illumina HiSeq2500 sequencer<b>Data format: </b>Filtered fastA files<b>Experimental factors: </b>Complexity-reduced genotyping by sequencing using three complexity reduction methods on bacterial isolates<b>Experimental features:</b> Combinations of enzymes of <i>Pst</i>I with <i>Mse</i>I and <i>Pst</i>I with <i>Hpa</i>II were used.<b>Data source location: </b>Australia.<br><b>1. Data:</b>These short-read fastA files are complexity-reduced genotyping data obtained from thermophilic bacterial isolates identified in samples from domestic hot water systems in the Australian Capital Territory (ACT), commercial compost available in the ACT and artesian bores in the Great Artesian Basin in South Australia. Samples were inoculated in LB agar plates and incubated at 62.2 degree Celsius, followed by DNA extraction with the method of chloroform-isoamyl alcohol solution (24:1), washed with Ethanol (70%) and dissolved in 10 mM Tris-Cl, pH 8.5. This was followed by digestion with selected pairs of restriction enzymes (<i>Pst</i>I with <i>Mse</i>I and <i>Pst</i>I with <i>Hpa</i>II), PCR amplification and sequencing using an Illumina HiSeq2500 sequencer. The number of reads for these sequences was approximately 150,000.<br><br><b>2. Experimental design, materials and methods.</b><b>2.1 Bacterial isolates</b>A total of 99 bacterial isolates were obtained from 27 different sampling sources. DNA extractions were performed in all bacterial isolates using the chloroform-isoamyl alcohol method as described by Talamantes-Becerra <i>et al.</i>, 2019.<br><b>2.2 Library preparation and sequencing </b>The library preparation followed the DArTseq™ (Canberra, Australia) DNA digestion method that uses restriction enzymes. The pairs of enzymes used were <i>Pst</i>I (5'-CTGCA|G-3') with <i>Mse</i>I (5'-TTA|A-3') and<i> Pst</i>I (5'-CTGCA|G-3') with <i>Hpa</i>II (5'-CCG|G-3'). Clustering and sequencing were done according to the Illumina (San Diego CA, US) protocols for the HiSeq Cluster Kit V4 recipe v9.0, HiSeq SR Flow Cell v4 and sequenced in a HiSeq 2500 for a total of 77 cycles. The reads obtained per sample were approximately 150,000.<br><br><b>2.3 Production of data files</b>The raw data obtained as fastQ files was processed using the DArTseq™ primary data processing pipeline for demultiplexing. The filtering uses stringency values of Phred score of 30, pass percentage 75 for the barcode and a minimum of Phred score of 10, pass percentage 50 for the whole-read as described by Georges<i> et al.</i>, 2018. Barcode adapters were trimmed and fragments of less than 29 bp were removed. The final length of unique fragments was between 30 to 69 bp.<br><b>Acknowledgements:</b>We would like to thank Dr M.A.(Rien) Habermehl for sharing his expertise and advice during the selection of sampling sites and indicating the best season for collecting samples in the Great Artesian Basin. We would like to thank D. Shrestha for collecting and sending back to us, mud and water samples from Birdsville and Stoney crossing artesian water bores. We would like to thank the station managers from the Birdsville Track for allowing us to collect samples from their artesian water bores. The author BTB, wishes to acknowledge Consejo Nacional de Ciencia y Tecnología (CONACYT) for providing a scholarship “Becas CONACYT al extranjero 2015” to pursue postgraduate studies. Genome sequencing was provided by MicrobesNG (http://www.microbesng.uk), which is supported by the BBSRC (grant number BB/L024209/1). We thank Distinguished Prof. Arthur Georges, Dr Andrzej Kilian for their valuable contributions. We also thank Dr Michelle Gahan and Prof. Dennis McNevin for their suggestions on project development and methods and for co-supervising the PhD project from which this work arises.<br>