Metatranscriptomics capabilities, discoveries, challenges ... metagenome genome gene metatranscriptome transcriptome transcript metaproteome proteome protein molecule cell environment metagenome genome gene metatranscriptome transcriptome transcript metaproteome proteome protein molecule cell environment Fractions of RNA rRNA (constitute upward to 90% of all RNA) → taxonomy mRNA→ genes expression regulatory ncRNA → role in metabolism (iron metabolism, virulence and quorum sensing, and to adapt quickly to changing environments) What information metatranscriptomics data give us? (a few examples) ? ? ? ? Anique Stecher, Stefan Neuhaus, Benjamin Lange, Stephan Frickenhaus, Bánk Beszteri, Peter G. Kroth & Klaus Valentin (2016) rRNA and rDNA based assessment of sea ice protist biodiversity from the central Arctic Ocean, European Journal of Phycology, 51:1, 31-46, DOI: 10.1080/09670262.2015.1077395 Anique Stecher, Stefan Neuhaus, Benjamin Lange, Stephan Frickenhaus, Bánk Beszteri, Peter G. Kroth & Klaus Valentin (2016) rRNA and rDNA based assessment of sea ice protist biodiversity from the central Arctic Ocean, European Journal of Phycology, 51:1, 31-46, DOI: 10.1080/09670262.2015.1077395 Important information: 1)Inflammatory bowel disease (IBD) is a group of chronic diseases of the digestive tract. One of those diseases is Crohn’s disease (CD) 2)Alistipes putredinis and Bacteroides vulgatus are both able to perform the methylerythritol phosphate (MEP) pathway, which indirectly induces acute inflammation. Schirmer, Melanie et al. “Dynamics of metatranscription in the inflammatory bowel disease gut microbiome.” Nature microbiology vol. 3,3 (2018): 337-346. doi:10.1038/s41564-017- 0089-z Bacterioides vulgatus Alistipes putredinis Schirmer, Melanie et al. “Dynamics of metatranscription in the inflammatory bowel disease gut microbiome.” Nature microbiology vol. 3,3 (2018): 337-346. doi:10.1038/s41564-017- 0089-z How plankton evolves, interconnects and moves? How are micro-organisms Tara oceans distributed in the oceans? How bacteria, plankton and viruses are linked? What influence does temperature, salinity, acidity and physico- chemical parameters? about 13Tb deposed in EBI ready to be analysed! https://oceans.taraexpeditions.org/en/m/about-tara/les-expeditions/tara-oceans/ Metatranscriptomics study of all transcriptomes in environment Which organism is active? How microorganisms and viruses interact? Which organism is responsible How microorganisms interact with each for producing particular other and with multicellular organisms? metabolite(s)? How gut microbiota affects How organisms react to different our health? conditions? Popularność badań Shakya M, Lo CC and Chain PSG (2019) Advances and Challenges in Metatranscriptomic Analysis. Front. Genet. 10:904. doi: 10.3389/fgene.2019.00904 drawbacks and problems difficulties to capture the entire metatranscriptome the short half-life of RNA a number of the technology-specific limitations a lot of data and time-consuming computer analysis the lack of metagenome and reference genomes What? metatranscriptomics Effect metagenomics metaproteomics Who? Zhang, X., Li, L., Butcher, J. et al. Advancing functional and translational microbiome research using meta-omics approaches. Microbiome 7, 154 (2019). https://doi.org/10.1186/s40168-019- 0767-6 Combining metagenomic and metatranscriptomics data which organisms are active and which are inactive co-assembling metagenomes and metatranscriptomes – more reads more accurate binning MetaTrans s Dark blue and turquoise- e quality check n Green – sorting rna i l Pink – functional analysis e Blue – taxonomy assignment p i P : Martinez, X. et al. MetaTrans: an open-source pipeline for metatranscriptomics. Sci. Rep. 6, 26447; doi: 10.1038/srep26447 (2016). Pipelines 8 analyses. Genome Biol 17, 260 (2016). https://doi.org/10.1186/s13059-016-1116- (2016). Biol 17, 260 analyses. Genome reference-independent integrated metagenomic and metatranscriptomic metatranscriptomic and metagenomic integrated reference-independent Narayanasamy, S., Jarosz, Y., Muller, E.E.L. et al. IMP: a pipeline for reproducible for reproducible pipeline a al. IMP: E.E.L. et Muller, Y., Jarosz, S., Narayanasamy, IMP – Combining Combining – IMP MT and MG and MT Bioinformatic Pipelines ● The are several pipelines: MetaTrans, IMP, SAMSA2, MOSCA, MetaQUBIC, FMAP … … but most of them have similar steps Do it yourself (in dry lab)! taxonomy separation checking transcriptom characterization of rRNA and quality assembly (binning) other RNA obtaining functional expression characterization and of unigenes abundance of unigenes Challenges deposing data with detailed metadata (with metadata, ideally with metagenome) development of long-read sequencing technologies improving pipelines and particular bioinformatics tools Bibliography Martinez, X., Pozuelo, M., Pascal, V. et al. MetaTrans: an open-source pipeline for metatranscriptomics. Sci Rep 6, 26447 (2016). https://doi.org/10.1038/srep26447 Kim, J., Kim, M.S., Koh, A.Y. et al. FMAP: Functional Mapping and Analysis Pipeline for metagenomics and metatranscriptomics studies. BMC Bioinformatics 17, 420 (2016). https://doi.org/10.1186/s12859-016-1278-0 Paul Saary, Alex L. Mitchell, Robert D. Finn Estimating the quality of eukaryotic genomes recovered from metagenomic analysis BioRxiv 2019.12.19.882753; doi: https://doi.org/10.1101/2019.12.19.882753 Sunagawa S, Karsenti E, Bowler C, Bork P. Computational eco-systems biology in Tara Oceans: translating data into knowledge. Mol Syst Biol. 2015;11(5):809. Published 2015 May 21. doi:10.15252/msb.20156272 Shakya M, Lo CC and Chain PSG (2019) Advances and Challenges in Metatranscriptomic Analysis. Front. Genet. 10:904. doi: 10.3389/fgene.2019.00904 Westreich ST, Treiber ML, Mills DA, Korf I, Lemay DG. SAMSA2: a standalone metatranscriptome analysis pipeline. BMC Bioinformatics. 2018;19(1):175. Published 2018 May 21. doi:10.1186/s12859-018-2189-z https://www.cd-genomics.com/the-principles-workflow-and-applications-of-metatranscriptomic-sequencing.htm https://galaxyproject.github.io/training-material/topics/metagenomics/tutorials/metatranscriptomics/tutorial.html .