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      Metagenomics versus total RNA sequencing: most accurate data-processing tools, microbial identification accuracy and perspectives for ecological assessments

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          Abstract

          Metagenomics and total RNA sequencing (total RNA-Seq) have the potential to improve the taxonomic identification of diverse microbial communities, which could allow for the incorporation of microbes into routine ecological assessments. However, these target-PCR-free techniques require more testing and optimization. In this study, we processed metagenomics and total RNA-Seq data from a commercially available microbial mock community using 672 data-processing workflows, identified the most accurate data-processing tools, and compared their microbial identification accuracy at equal and increasing sequencing depths. The accuracy of data-processing tools substantially varied among replicates. Total RNA-Seq was more accurate than metagenomics at equal sequencing depths and even at sequencing depths almost one order of magnitude lower than those of metagenomics. We show that while data-processing tools require further exploration, total RNA-Seq might be a favorable alternative to metagenomics for target-PCR-free taxonomic identifications of microbial communities and might enable a substantial reduction in sequencing costs while maintaining accuracy. This could be particularly an advantage for routine ecological assessments, which require cost-effective yet accurate methods, and might allow for the incorporation of microbes into ecological assessments.

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          Trimmomatic: a flexible trimmer for Illumina sequence data

          Motivation: Although many next-generation sequencing (NGS) read preprocessing tools already existed, we could not find any tool or combination of tools that met our requirements in terms of flexibility, correct handling of paired-end data and high performance. We have developed Trimmomatic as a more flexible and efficient preprocessing tool, which could correctly handle paired-end data. Results: The value of NGS read preprocessing is demonstrated for both reference-based and reference-free tasks. Trimmomatic is shown to produce output that is at least competitive with, and in many cases superior to, that produced by other tools, in all scenarios tested. Availability and implementation: Trimmomatic is licensed under GPL V3. It is cross-platform (Java 1.5+ required) and available at http://www.usadellab.org/cms/index.php?page=trimmomatic Contact: usadel@bio1.rwth-aachen.de Supplementary information: Supplementary data are available at Bioinformatics online.
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            The Sequence Alignment/Map format and SAMtools

            Summary: The Sequence Alignment/Map (SAM) format is a generic alignment format for storing read alignments against reference sequences, supporting short and long reads (up to 128 Mbp) produced by different sequencing platforms. It is flexible in style, compact in size, efficient in random access and is the format in which alignments from the 1000 Genomes Project are released. SAMtools implements various utilities for post-processing alignments in the SAM format, such as indexing, variant caller and alignment viewer, and thus provides universal tools for processing read alignments. Availability: http://samtools.sourceforge.net Contact: rd@sanger.ac.uk
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              Fast gapped-read alignment with Bowtie 2.

              As the rate of sequencing increases, greater throughput is demanded from read aligners. The full-text minute index is often used to make alignment very fast and memory-efficient, but the approach is ill-suited to finding longer, gapped alignments. Bowtie 2 combines the strengths of the full-text minute index with the flexibility and speed of hardware-accelerated dynamic programming algorithms to achieve a combination of high speed, sensitivity and accuracy.
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                Author and article information

                Contributors
                Journal
                Nucleic Acids Res
                Nucleic Acids Res
                nar
                Nucleic Acids Research
                Oxford University Press
                0305-1048
                1362-4962
                09 September 2022
                18 August 2022
                18 August 2022
                : 50
                : 16
                : 9279-9293
                Affiliations
                Department of Integrative Biology, University of Guelph , Guelph, ON N1G 2W1, Canada
                Centre for Biodiversity Genomics, University of Guelph , Guelph, ON N1G 2W1, Canada
                Department of Integrative Biology, University of Guelph , Guelph, ON N1G 2W1, Canada
                Department of Integrative Biology, University of Guelph , Guelph, ON N1G 2W1, Canada
                SHARCNET, University of Guelph , Guelph, ON N1G 2W1, Canada
                Department of Integrative Biology, University of Guelph , Guelph, ON N1G 2W1, Canada
                Department of Integrative Biology, University of Guelph , Guelph, ON N1G 2W1, Canada
                Centre for Biodiversity Genomics, University of Guelph , Guelph, ON N1G 2W1, Canada
                Author notes
                To whom correspondence should be addressed. Tel: +1 519 824 4120; Fax: +1 519 824 5703; Email: hempelc@ 123456uoguelph.ca
                Author information
                https://orcid.org/0000-0002-2324-3115
                https://orcid.org/0000-0002-8992-575X
                Article
                gkac689
                10.1093/nar/gkac689
                9458450
                35979944
                2c02b022-b2fd-4529-9d49-78755f848c95
                © The Author(s) 2022. Published by Oxford University Press on behalf of Nucleic Acids Research.

                This is an Open Access article distributed under the terms of the Creative Commons Attribution-NonCommercial License ( https://creativecommons.org/licenses/by-nc/4.0/), which permits non-commercial re-use, distribution, and reproduction in any medium, provided the original work is properly cited. For commercial re-use, please contact journals.permissions@ 123456oup.com

                History
                : 29 July 2022
                : 05 July 2022
                : 03 June 2022
                Page count
                Pages: 15
                Funding
                Funded by: Canada First Research Excellence Fund, DOI 10.13039/501100010785;
                Funded by: University of Guelph, DOI 10.13039/100008986;
                Categories
                AcademicSubjects/SCI00010
                Genomics

                Genetics
                Genetics

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