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      Integrating multi-platform assembly to recover MAGs from hot spring biofilms: insights into microbial diversity, biofilm formation, and carbohydrate degradation

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          Abstract

          Background

          Hot spring biofilms provide a window into the survival strategies of microbial communities in extreme environments and offer potential for biotechnological applications. This study focused on green and brown biofilms thriving on submerged plant litter within the Sungai Klah hot spring in Malaysia, characterised by temperatures of 58–74 °C. Using Illumina shotgun metagenomics and Nanopore ligation sequencing, we investigated the microbial diversity and functional potential of metagenome-assembled genomes (MAGs) with specific focus on biofilm formation, heat stress response, and carbohydrate catabolism.

          Results

          Leveraging the power of both Illumina short-reads and Nanopore long-reads, we employed an Illumina-Nanopore hybrid assembly approach to construct MAGs with enhanced quality. The dereplication process, facilitated by the dRep tool, validated the efficiency of the hybrid assembly, yielding MAGs that reflected the intricate microbial diversity of these extreme ecosystems. The comprehensive analysis of these MAGs uncovered intriguing insights into the survival strategies of thermophilic taxa in the hot spring biofilms. Moreover, we examined the plant litter degradation potential within the biofilms, shedding light on the participation of diverse microbial taxa in the breakdown of starch, cellulose, and hemicellulose. We highlight that Chloroflexota and Armatimonadota MAGs exhibited a wide array of glycosyl hydrolases targeting various carbohydrate substrates, underscoring their metabolic versatility in utilisation of carbohydrates at elevated temperatures.

          Conclusions

          This study advances understanding of microbial ecology on plant litter under elevated temperature by revealing the functional adaptation of MAGs from hot spring biofilms. In addition, our findings highlight potential for biotechnology application through identification of thermophilic lignocellulose-degrading enzymes. By demonstrating the efficiency of hybrid assembly utilising Illumina-Nanopore reads, we highlight the value of combining multiple sequencing methods for a more thorough exploration of complex microbial communities.

          Supplementary Information

          The online version contains supplementary material available at 10.1186/s40793-024-00572-7.

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          Most cited references57

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          Cutadapt removes adapter sequences from high-throughput sequencing reads

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            CheckM: assessing the quality of microbial genomes recovered from isolates, single cells, and metagenomes

            Large-scale recovery of genomes from isolates, single cells, and metagenomic data has been made possible by advances in computational methods and substantial reductions in sequencing costs. Although this increasing breadth of draft genomes is providing key information regarding the evolutionary and functional diversity of microbial life, it has become impractical to finish all available reference genomes. Making robust biological inferences from draft genomes requires accurate estimates of their completeness and contamination. Current methods for assessing genome quality are ad hoc and generally make use of a limited number of “marker” genes conserved across all bacterial or archaeal genomes. Here we introduce CheckM, an automated method for assessing the quality of a genome using a broader set of marker genes specific to the position of a genome within a reference genome tree and information about the collocation of these genes. We demonstrate the effectiveness of CheckM using synthetic data and a wide range of isolate-, single-cell-, and metagenome-derived genomes. CheckM is shown to provide accurate estimates of genome completeness and contamination and to outperform existing approaches. Using CheckM, we identify a diverse range of errors currently impacting publicly available isolate genomes and demonstrate that genomes obtained from single cells and metagenomic data vary substantially in quality. In order to facilitate the use of draft genomes, we propose an objective measure of genome quality that can be used to select genomes suitable for specific gene- and genome-centric analyses of microbial communities.
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              MEGAHIT: an ultra-fast single-node solution for large and complex metagenomics assembly via succinct de Bruijn graph.

              MEGAHIT is a NGS de novo assembler for assembling large and complex metagenomics data in a time- and cost-efficient manner. It finished assembling a soil metagenomics dataset with 252 Gbps in 44.1 and 99.6 h on a single computing node with and without a graphics processing unit, respectively. MEGAHIT assembles the data as a whole, i.e. no pre-processing like partitioning and normalization was needed. When compared with previous methods on assembling the soil data, MEGAHIT generated a three-time larger assembly, with longer contig N50 and average contig length; furthermore, 55.8% of the reads were aligned to the assembly, giving a fourfold improvement. © The Author 2015. Published by Oxford University Press. All rights reserved. For Permissions, please e-mail: journals.permissions@oup.com.
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                Author and article information

                Contributors
                rajesh.sani@sdsmt.edu
                gohkianmau@utm.my
                Journal
                Environ Microbiome
                Environ Microbiome
                Environmental Microbiome
                BioMed Central (London )
                2524-6372
                6 May 2024
                6 May 2024
                2024
                : 19
                : 29
                Affiliations
                [1 ]Codon Genomics, 42300 Seri Kembangan, Selangor, Malaysia
                [2 ]Faculty of Science, Universiti Teknologi Malaysia, ( https://ror.org/026w31v75) 81310 Skudai, Johor, Malaysia
                [3 ]School of Professional and Continuing Education, Universiti Teknologi Malaysia, ( https://ror.org/026w31v75) 81310 Skudai, Johor, Malaysia
                [4 ]Division of Genetics and Molecular Biology, Institute of Biological Sciences, Faculty of Science, University of Malaya, ( https://ror.org/00rzspn62) Kuala Lumpur, Malaysia
                [5 ]Department of Biological Sciences, National University of Singapore, ( https://ror.org/01tgyzw49) Singapore, Singapore
                [6 ]Department of Chemical and Biological Engineering, South Dakota School of Mines and Technology, ( https://ror.org/00ch7yk27) Rapid City, SD 57701 USA
                Article
                572
                10.1186/s40793-024-00572-7
                11071339
                38706006
                4fbe8f4f-8ec2-48e3-b637-5a4768029954
                © The Author(s) 2024

                Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/. The Creative Commons Public Domain Dedication waiver ( http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated in a credit line to the data.

                History
                : 22 November 2023
                : 22 April 2024
                Funding
                Funded by: Malaysia Fundamental Research Grant Scheme (FRGS)
                Award ID: FRGS/1/2023/STG02/UTM/02/1, FRGS/1/2019/STG03/UTM/02/1, FRGS/1/2019/STG04/UTM/02/4
                Award ID: FRGS/1/2023/STG02/UTM/02/1, FRGS/1/2019/STG03/UTM/02/1, FRGS/1/2019/STG04/UTM/02/4
                Award ID: FRGS/1/2023/STG02/UTM/02/1, FRGS/1/2019/STG03/UTM/02/1, FRGS/1/2019/STG04/UTM/02/4
                Award ID: FRGS/1/2023/STG02/UTM/02/1, FRGS/1/2019/STG03/UTM/02/1, FRGS/1/2019/STG04/UTM/02/4
                Award ID: FRGS/1/2023/STG02/UTM/02/1, FRGS/1/2019/STG03/UTM/02/1, FRGS/1/2019/STG04/UTM/02/4
                Award Recipient :
                Funded by: UTM QuickWin grant
                Award ID: 4J549
                Award ID: 4J549
                Award Recipient :
                Funded by: Singapore Ministry of Education ARC Tier 2 fund
                Award ID: T2EP30123-0028
                Award Recipient :
                Funded by: FundRef http://dx.doi.org/10.13039/100000001, National Science Foundation;
                Award ID: 1736255, 1849206, and 1920954
                Award Recipient :
                Categories
                Research
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                © BioMed Central Ltd., part of Springer Nature 2024

                hybrid assembly,lignocellulose degradation,metagenome-assembled genomes,microbial mat,thermophile

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