{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2024,3,3]],"date-time":"2024-03-03T00:18:22Z","timestamp":1709425102690},"reference-count":28,"publisher":"F1000 Research Ltd","license":[{"start":{"date-parts":[[2021,2,11]],"date-time":"2021-02-11T00:00:00Z","timestamp":1613001600000},"content-version":"tdm","delay-in-days":0,"URL":"http:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"funder":[{"DOI":"10.13039\/100000001","name":"National Science Foundation","doi-asserted-by":"publisher","award":["1458524"],"id":[{"id":"10.13039\/100000001","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100000054","name":"National Cancer Institute","doi-asserted-by":"publisher","award":["1U24CA199347"],"id":[{"id":"10.13039\/100000054","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/100007249","name":"University of Minnesota","doi-asserted-by":"crossref","id":[{"id":"10.13039\/100007249","id-type":"DOI","asserted-by":"crossref"}]}],"content-domain":{"domain":["f1000research.com"],"crossmark-restriction":false},"short-container-title":["F1000Res"],"abstract":"The Human Microbiome Project (HMP) aided in understanding the role of microbial communities and the influence of collective genetic material (the \u2018microbiome\u2019) in human health and disease. With the evolution of new sequencing technologies, researchers can now investigate the microbiome and map its influence on human health. Advances in bioinformatics methods for next-generation sequencing (NGS) data analysis have helped researchers to gain an in-depth knowledge about the taxonomic and genetic composition of microbial communities. Metagenomic-based methods have been the most commonly used approaches for microbiome analysis; however, it primarily extracts information about taxonomic composition and genetic potential of the microbiome under study, lacking quantification of the gene products (RNA and proteins). Conversely, metatranscriptomics, the study of a microbial community\u2019s RNA expression, can reveal the dynamic gene expression of individual microbial populations and the community as a whole, ultimately providing information about the active pathways in the microbiome.\u00a0 In order to address the analysis of NGS data, the ASaiM analysis framework was previously developed and made available via the Galaxy platform. Although developed for both metagenomics and metatranscriptomics, the original publication demonstrated the use of ASaiM only for metagenomics, while thorough testing for metatranscriptomics data was lacking.\u00a0 In the current study, we have focused on validating and optimizing the tools within ASaiM for metatranscriptomics data. As a result, we deliver a robust workflow that will enable researchers to understand dynamic functional response of the microbiome in a wide variety of metatranscriptomics studies. This improved and optimized ASaiM-metatranscriptomics (ASaiM-MT) workflow is publicly available via the ASaiM framework, documented and supported with training material so that users can interrogate and characterize metatranscriptomic data, as part of larger meta-omic studies of microbiomes.<\/ns4:p>","DOI":"10.12688\/f1000research.28608.1","type":"journal-article","created":{"date-parts":[[2021,2,11]],"date-time":"2021-02-11T19:30:00Z","timestamp":1613071800000},"page":"103","update-policy":"http:\/\/dx.doi.org\/10.12688\/f1000research.crossmark-policy","source":"Crossref","is-referenced-by-count":1,"title":["ASaiM-MT: a validated and optimized ASaiM workflow for metatranscriptomics analysis within Galaxy framework"],"prefix":"10.12688","volume":"10","author":[{"ORCID":"http:\/\/orcid.org\/0000-0001-9818-0537","authenticated-orcid":false,"given":"Subina","family":"Mehta","sequence":"first","affiliation":[]},{"given":"Marie","family":"Crane","sequence":"additional","affiliation":[]},{"given":"Emma","family":"Leith","sequence":"additional","affiliation":[]},{"ORCID":"http:\/\/orcid.org\/0000-0001-9852-1987","authenticated-orcid":false,"given":"B\u00e9r\u00e9nice","family":"Batut","sequence":"additional","affiliation":[]},{"given":"Saskia","family":"Hiltemann","sequence":"additional","affiliation":[]},{"given":"Magnus \u00d8","family":"Arntzen","sequence":"additional","affiliation":[]},{"given":"Benoit J.","family":"Kunath","sequence":"additional","affiliation":[]},{"given":"Francesco","family":"Delogu","sequence":"additional","affiliation":[]},{"given":"Ray","family":"Sajulga","sequence":"additional","affiliation":[]},{"given":"Praveen","family":"Kumar","sequence":"additional","affiliation":[]},{"given":"James E.","family":"Johnson","sequence":"additional","affiliation":[]},{"ORCID":"http:\/\/orcid.org\/0000-0001-6801-2559","authenticated-orcid":false,"given":"Timothy J.","family":"Griffin","sequence":"additional","affiliation":[]},{"ORCID":"http:\/\/orcid.org\/0000-0003-0984-0973","authenticated-orcid":false,"given":"Pratik D.","family":"Jagtap","sequence":"additional","affiliation":[]}],"member":"2560","published-online":{"date-parts":[[2021,2,11]]},"reference":[{"key":"ref-1","doi-asserted-by":"publisher","first-page":"1-14","DOI":"10.1007\/s00394-018-1703-4","article-title":"The role of the microbiome for human health: from basic science to clinical applications.","volume":"57","author":"M Mohajeri","year":"2018","journal-title":"Eur J Nutr."},{"key":"ref-2","doi-asserted-by":"publisher","first-page":"1091","DOI":"10.1038\/s41587-019-0252-6","article-title":"Author Correction: Reproducible, interactive, scalable and extensible microbiome data science using QIIME 2. 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with reservations","URL":"https:\/\/f1000research.com\/articles\/10-103\/v1#article-reports","order":0,"name":"referee-status","label":"Referee status","group":{"name":"current-referee-status","label":"Current Referee Status"}},{"value":"10.5256\/f1000research.31653.r79463, Caitlin Simopoulos, Department of Biochemistry, Microbiology and Immunology, University of Ottawa, Ottawa, ON, Canada, 18 Feb 2021, version 1, 1 approved with reservations","URL":"https:\/\/f1000research.com\/articles\/10-103\/v1#referee-response-79463","order":0,"name":"referee-response-79463","label":"Referee Report","group":{"name":"article-reports","label":"Article Reports"}},{"value":"Subina Mehta<\/b>; \nPosted: 25 Mar 2021<\/i>; We greatly appreciate the reviewer\u2019s comments and suggestions. We have incorporated the required changes in the updated version of the manuscript. \nMajor comments:<\/b><\/u> \n1. In your discussion, you highlighted a major limitation of ASaiM-MT: the fact that it can only handle a single sample and does not complete comparative analysis. However, you also mention that you have developed post-processing tools for this reason. It might be good to highlight that ASaiM-MT is essential (in particular for data pre-processing and identification of functional and taxonomic information) to be completed before statistical analysis. In addition, is it possible to process data in ASaiM-MT in batches? This might also lessen the perceived limitation of the tool.<\/b> \nA: <\/b>\nWe would like to thank the reviewer for the comment. We have edited the abstract and mentioned that ASaiM-MT is essential for preprocessing and identification of taxonomy and functional information before performing comparative analysis. Also, Galaxy has a function of providing datasets as a collection rather than a single input which can be used to handle multiple datasets. In the Galaxy platform, we have other software tools such as MT2MQ and metaQuantome, which can perform comparative statistical analysis.<\/i> \n2. Sometimes it is not completely clear why ASaiM-MT is needed if ASaiM was developed for both meta-genomics and -transcriptomics. It is important to be specific and highlight the rationale for developing the ASaiM-MT workflow. An example: \u201cFor performing this action, the original ASaiM Shotgun workflow used the FASTQ-joiner to join the reads. However, in the ASaiM-MT version, we use the FASTQ interlacer. FASTQ interlacer joins the forward (\/1) and the reverse reads (\/2) using the sequence identifiers; sequences without designation will be named as single reads. The reason ASaiM-MT uses FASTQ-interlacer rather than FASTQ-joiner is because the joiner tool combines the forward and reverse read sequence together while the interlacer puts the forward and reverse read sequences in the same file while retaining the entity of each read along with an additional file with unpaired sequences.\u201d Was this change made because FASTQ interlacer is more appropriate for RNA sequencing, or was it a change to improve on the original ASaiM workflow?<\/b> \nA: <\/b>\nThank you for this comment. We replaced the FASTQ-joiner tool with the FASTQ interlacer to improve on the original workflow. The FASTQ interlacer tool maintains the integrity of reads by maintaining the forward and the reverse sequence identifiers, as compared to joining them into a single read file, as is done by FASTQ-joiner tool.<\/i> \n3. The abstract and introduction seem to emphasize the importance of studying the human microbiome and its connections to health and well-being, however, this manuscript uses a microbial community obtained from a biogas reactor. This is a very interesting community that warrants further research, but the introduction does not match the sample of interest. I suggest updating the introduction to focus less on the human microbiome and emphasize the importance of studying microbial communities in general. Alternatively, if you\u2019d like to focus on human microbiomes, the example data used should reflect the human focus.<\/b> \nA: <\/b>\nWe thank the reviewer for the comment and have edited the abstract and introduction to highlight the importance of microbiome research in ecology as well as clinical research.<\/i> \n <\/b> \n4. In your discussion, you say: \u201cThere are a few tools that can be used alternatively or in addition to the existing tools.\u201d Does this mean that there are other tools like ASaiM-MT that can be used? Your introduction mainly listed tools that complete specific tasks and not an end to end workflow. If there are other workflow tools, a comparison should be highlighted in the discussion or introduction. What makes ASaiM-MT unique compared to other tools\/workflows? What are its strengths? Is it more user-friendly than other tools? Expanding on this will help strengthen your conclusions about the tool itself.<\/b> \nA: <\/b>\nWe thank the reviewer for the comment. The tools mentioned in the discussion offer an alternative option to the tools that we have used in the AsaiM-MT workflow, especially if the users have a preference. However, although we consider these tools to be appropriate to metatranscriptomics research, we have not tested these tools. ASaiM-MT workflow, due to its availability in Galaxy, offers an user-friendly option to the existing command line tools. The ASaiM-MT workflow has been tested with different datasets to ensure its compatibility. There is a systematic documentation available for the usage of the workflow in the Galaxy Training Network (GTN). Users can also ask questions to developers and users via the Gitter channel, if needed.<\/i> \n <\/b> \nMinor comments:<\/b><\/u> \n1. There are sections that can read like a \u201cgrocery list\u201d of software names (eg. second paragraph of the introduction). I understand why you are listing them, but it could be useful to emphasize that each of these tools performs a single task and need to be put together into a workflow for a complete experiment. These tools are also often missing citations.<\/b> \nA: <\/b>\nThanks to the reviewer for the comment. The tools listed are alternatives to the existing tools in the workflow and have not been incorporated as a workflow. We have added citations to these tools.<\/i> \n <\/b> \n2. It can be difficult to understand which words are actually software names. Is it possible to type them in a monospace font? Or to bold the names?<\/b> \nA: <\/b>\nThanks to the reviewer for this comment. I have formatted the tool names by making them italic.<\/i> \n3. At times the \u201cMethods\u201d section can get confusing, particularly when MT is being compared to the original ASaiM. My suggestion is to include numbers or roman numerals in the \u201cin between\u201d steps as described in Figure 2. That way you can also reference them in the text. For example, in \u201ca) Preprocessing\u201d there would be i. Input files, ii. Quality Control, iii. Adapter Trimming\u2026 etc.. It might also be useful to include these headers (and numbers\/letters) in your Methods section to let readers follow along.<\/b> \nA: <\/b>\nThank you for the comment. I have reformatted the method section according to Figure 2.<\/i> \n <\/b> \n4. \u201cPMID 30298254\u201d is used instead of a citation<\/b> \nA: <\/b>\nThank you reviewer for pointing this out. I have made the required change.<\/i>","URL":"https:\/\/f1000research.com\/articles\/10-103\/v1#referee-comment-6496","order":1,"name":"referee-comment-6496","label":"Referee Comment","group":{"name":"article-reports","label":"Article Reports"}},{"value":"We acknowledge funding for this work from the grant National Cancer Institute - Informatics Technology for Cancer Research (NCI-ITCR) grant 1U24CA199347, National Science Foundation (U.S.) grant 1458524 to T.J.G and a grant through the Norwegian Centennial Chair (NOCC) program at the University of Minnesota to T.J.G and M.A. The European Galaxy server that was used for data analysis is in part funded by Collaborative Research Centre 992 Medical Epigenetics (DFG grant SFB 992\/1 2012) and German Federal Ministry of Education and Research (BMBF grants 031 A538A\/A538C RBC, 031L0101B\/031L0101C de.NBI-epi, 031L0106 de.STAIR (de.NBI)). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.","order":2,"name":"grant-information","label":"Grant Information"},{"value":"This is an open access article distributed under the terms of the Creative Commons Attribution Licence, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.","order":0,"name":"copyright-info","label":"Copyright"}]}}