ONTOLOGY SOURCE REFERENCE
Term Source Name	EDAM	NCBITAXON	MTBLS	EFO	OBI	CHMO	
Term Source File			https://www.ebi.ac.uk/metabolights/		http://data.bioontology.org/ontologies/OBI		http://purl.obolibrary.org/obo/ncit.owl
Term Source Version		2020-09-11	1.0		29		18.10e
Term Source Description			Metabolights Ontology		Ontology for Biomedical Investigations		NCI Thesaurus OBO Edition
INVESTIGATION
Investigation Identifier	MTBLS2060
Investigation Title	Investigation
Investigation Description	Created using the MetaboLights Online Editor (MOE)
Investigation Submission Date	2020-09-10
Investigation Public Release Date	2021-08-05
Comment[Created With Configuration]	MetaboLightsConfig20150707
Comment[Last Opened With Configuration]	MetaboLightsConfig20150707
INVESTIGATION PUBLICATIONS
Investigation PubMed ID
Investigation Publication DOI
Investigation Publication Author List
Investigation Publication Title
Investigation Publication Status
Investigation Publication Status Term Accession Number
Investigation Publication Status Term Source REF
INVESTIGATION CONTACTS
Investigation Person Last Name
Investigation Person First Name
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Investigation Person Email
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Investigation Person Fax
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Investigation Person Affiliation
Investigation Person Roles
Investigation Person Roles Term Accession Number
Investigation Person Roles Term Source REF
STUDY
Study Identifier	MTBLS2060
Study Title	Prediction of complex phenotypes using the Drosophila melanogaster metabolome
Study Description	Understanding the genotype-phenotype map and how variation at different levels of biological organization is associated are central topics in modern biology. Fast developments in sequencing technologies and other molecular omic tools enable researchers to obtain detailed information on variation at DNA level and on intermediate endophenotypes, such as RNA, proteins and metabolites. This can facilitate our understanding of the link between genotypes and molecular and functional organismal phenotypes. Here, we use the Drosophila melanogaster Genetic Reference Panel and nuclear magnetic resonance (NMR) metabolomics to investigate the ability of the metabolome to predict organismal phenotypes. We performed NMR metabolomics on four replicate pools of male flies from each of 170 different isogenic lines. Our results show that metabolite profiles are variable among the investigated lines and that this variation is highly heritable. Second, we identify genes associated with metabolome variation. Third, using the metabolome gave better prediction accuracies than genomic information for four of five quantitative traits analyzed. Our comprehensive characterization of population-scale diversity of metabolomes and its genetic basis illustrates that metabolites have large potential as predictors of organismal phenotypes. This finding is of great importance, e.g., in human medicine, evolutionary biology and animal and plant breeding.
Study Submission Date	2020-09-10
Study Public Release Date	2021-08-05
Study File Name	s_MTBLS2060.txt
STUDY DESIGN DESCRIPTORS
Study Design Type	nuclear magnetic resonance spectroscopy	phenotype	untargeted metabolites	Drosophila Genetic Reference Panel
Study Design Type Term Accession Number	http://purl.obolibrary.org/obo/CHMO_0000591	http://www.ebi.ac.uk/efo/EFO_0000651	http://www.ebi.ac.uk/metabolights/ontology/MTBLS_000279	http://www.ebi.ac.uk/metabolights/ontology/placeholder
Study Design Type Term Source REF	CHMO	EFO	MTBLS	MTBLS
STUDY PUBLICATIONS
Study PubMed ID	33510469
Study Publication DOI	10.1038/s41437-021-00404-1
Study Publication Author List	Rohde PD, Kristensen TN, Sarup P, Muñoz J, Malmendal A.
Study Publication Title	Prediction of complex phenotypes using the Drosophila melanogaster metabolome.
Study Publication Status	Published
Study Publication Status Term Accession Number	http://www.ebi.ac.uk/efo/EFO_0001796
Study Publication Status Term Source REF	EFO
STUDY FACTORS
Study Factor Name	DGRP line
Study Factor Type	isogenic line
Study Factor Type Term Accession Number	http://www.ebi.ac.uk/metabolights/ontology/placeholder
Study Factor Type Term Source REF	MTBLS
STUDY ASSAYS
Study Assay File Name	a_MTBLS2060_NMR___metabolite_profiling.txt
Study Assay Measurement Type	metabolite profiling
Study Assay Measurement Type Term Accession Number	http://purl.obolibrary.org/obo/OBI_0000366
Study Assay Measurement Type Term Source REF	OBI
Study Assay Technology Type	NMR spectroscopy
Study Assay Technology Type Term Accession Number	http://purl.obolibrary.org/obo/OBI_0000623
Study Assay Technology Type Term Source REF	OBI
Study Assay Technology Platform	Nuclear Magnetic Resonance (NMR) -
STUDY PROTOCOLS
Study Protocol Name	Sample collection	Extraction	NMR sample	NMR spectroscopy	NMR assay	Data transformation	Metabolite identification
Study Protocol Type	Sample collection	Extraction	NMR sample	NMR spectroscopy	NMR assay	Data transformation	Metabolite identification
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Study Protocol Description	<p>We used 170 inbred lines of the DGRP<strong>[1][2]</strong>. The DGRP lines were established by 20 consecutive generations of full sibling inbreeding from isofemales collected at the farmer’s market in Raleigh, NC. Complete genome sequence of the DGRP lines have been obtained using Illumina platform, and is publicly available<strong>[1][2]</strong>. The DGRP lines were maintained on standard Drosophila medium<strong>[3]</strong> in a 23 °C climate chamber at 50% relative humidity with a 12-h:12-h light:dark cycle. For this study only male flies with an average age of 24 h were used. Flies were collected from 170 DGRP lines in 4 replicates containing 20 flies per replicate. Flies were snap frozen at 3 d of age and kept at –80 °C. </p><p><br></p><p><strong>Ref:</strong></p><p><strong>[1]</strong> Huang W, Massouras A, Inoue Y, Peiffer J, Ràmia M et al. Natural variation in genome architecture among 205 Drosophila melanogaster Genetic Reference Panel lines. Genome Res. 2014 Jul;24(7):1193-208. doi:10.1101/gr.171546.113. PMID:24714809</p><p><strong>[2]</strong> Mackay TF, Richards S, Stone EA, Barbadilla A, Ayroles JF et al. The Drosophila melanogaster Genetic Reference Panel. Nature. 2012 Feb 8;482(7384):173-8. doi:10.1038/nature10811. PMID:22318601. </p><p><strong>[3]</strong> Kristensen TN, Henningsen AK, Aastrup C, Bech-Hansen M, Bjerre LB et al. Fitness components of Drosophila melanogaster developed on a standard laboratory diet or a typical natural food source. Insect Sci. 2016 Oct;23(5):771-9. doi:10.1111/1744-7917.12239. PMID:25989059.</p>	<p>Samples were mechanically homogenized with a Kinematica, Pt 1200 (Buch &amp; Holm A/S, Herlev, Denmark) in 1 ml of ice-cold acetonitrile (50%) for 45 s. Hereafter samples were centrifuged (10,000 x g) for 10 min at 4 °C and the supernatant (900 μl) was transferred to new tubes, snap frozen and stored at –80 °C. The supernatant was lyophilized and stored at −80 °C.</p>	<p>Immediately before NMR measurements, samples were rehydrated in 200 ml of 50 mM phosphate buffer (pH 7.4) in D2O, and 180 μl was transferred to a 3 mm NMR tube. The buffer contained 50 mg/l of the chemical shift reference 2,2-Dimethyl-2-silapentane-5-sulfonate-d6, sodium salt (DSS), and 50 mg/l of sodium azide to prevent bacterial growth.</p>	<p>NMR measurements were performed at 25 °C on a Bruker Avance III HD 800 spectrometer (Bruker Biospin, Rheinstetten, Germany), operating at a 1H frequency of 799.87 MHz, and equipped with a 3 mm TCI cold probe.</p>	<p>1H NMR spectra were acquired using a standard NOESYPR1D experiment with a 100 ms delay. A total of 128 transients of 32 K data points spanning a spectral width of 20 ppm were collected.</p>	<p>The spectra were processed using <strong>Topspin</strong> (Bruker Biospin, Rheinstetten, Germany). An exponential line-broadening of 0.3 Hz was applied to the free-induction decay prior to Fourier transformation. All spectra were referenced to the DSS signal at 0 ppm, manually phased and baseline corrected. The spectra were aligned using <strong>icoshift[1]</strong>. The region around the residual water signal (4.87-4.70 ppm) was removed in order for the water signal not to interfere with the analysis. The high- and low-field ends of the spectrum, where no signals except the reference signal from DSS appear, were also removed (i.e., leaving data between 9.7 and 0.7 ppm). The spectra were normalized by <strong>probabilistic quotient area normalization[2]</strong>.</p><p><br></p><p><strong>Refs:</strong></p><p><strong>[1]</strong> Savorani F, Tomasi G, Engelsen SB. icoshift: A versatile tool for the rapid alignment of 1D NMR spectra. J Magn Reson. 2010 Feb;202(2):190-202. doi:10.1016/j.jmr.2009.11.012. PMID:20004603.</p><p><strong>[2]</strong> Dieterle F, Ross A, Schlotterbeck G, Senn H. Probabilistic quotient normalization as robust method to account for dilution of complex biological mixtures. Application in 1H NMR metabonomics. Anal Chem. 2006 Jul 1;78(13):4281-90. doi:10.1021/ac051632c. PMID:16808434.</p>	<p>Metabolite assignments were done based on chemical shifts, using earlier assignments and spectral databases previously described<strong>[1]-[5]</strong> together with <strong>Chenomx NMR Suite</strong> (Chenomx Inc).</p><p><br></p><p><strong>Refs:</strong></p><p><strong>[1]</strong> Malmendal A, Overgaard J, Bundy JG, Sørensen JG, Nielsen NC, Loeschcke V, Holmstrup M. Metabolomic profiling of heat stress: hardening and recovery of homeostasis in Drosophila. Am J Physiol Regul Integr Comp Physiol. 2006 Jul;291(1):R205-12. doi:10.1152/ajpregu.00867.2005. PMID:16469831.</p><p><strong>[2]</strong> Cui Q, Lewis IA, Hegeman AD, Anderson ME, Li J, Schulte CF, Westler WM, Eghbalnia HR, Sussman MR, Markley JL. Metabolite identification via the Madison Metabolomics Consortium Database. Nat Biotechnol. 2008 Feb;26(2):162-4. doi:10.1038/nbt0208-162. PMID:18259166.</p><p><strong>[3]</strong> Ulrich EL, Akutsu H, Doreleijers JF, Harano Y, Ioannidis YE, Lin J, Livny M, Mading S, Maziuk D, Miller Z, Nakatani E, Schulte CF, Tolmie DE, Kent Wenger R, Yao H, Markley JL. BioMagResBank. Nucleic Acids Res. 2008 Jan;36(Database issue):D402-8. doi:10.1093/nar/gkm957. PMID:17984079.</p><p><strong>[4]</strong> Schou WS, Ashina S, Amin FM, Goadsby PJ, Ashina M. Calcitonin gene-related peptide and pain: a systematic review. J Headache Pain. 2017 Dec;18(1):34. doi:10.1186/s10194-017-0741-2. PMID:28303458.</p><p><strong>[5]</strong> Wishart DS, Feunang YD, Marcu A, Guo AC, Liang K et al. HMDB 4.0: the human metabolome database for 2018. Nucleic Acids Res. 2018 Jan 4;46(D1):D608-D617. doi:10.1093/nar/gkx1089. PMID:29140435.</p>
Study Protocol URI							
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Study Protocol Parameters Name		Extraction Method	NMR tube type;Solvent;Sample pH;Temperature	Instrument;NMR Probe;Number of transients;Pulse sequence name;Magnetic field strength			
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STUDY CONTACTS
Study Person Last Name	Malmendal	Rohde	Kristensen	Sarup	Muñoz
Study Person First Name	Anders	Palle Duun	Torsten Nygaard	Pernille	Joaquin
Study Person Mid Initials					
Study Person Email	amalm@ruc.dk				
Study Person Phone					
Study Person Fax					
Study Person Address	Department of Science and Environment, Roskilde University, Roskilde, Denmark				
Study Person Affiliation	Roskilde University				
Study Person Roles	submitter	Author	Author	Author	Author
Study Person Roles Term Accession Number	http://www.ebi.ac.uk/efo/EFO_0001741	http://purl.obolibrary.org/obo/NCIT_C42781	http://purl.obolibrary.org/obo/NCIT_C42781	http://purl.obolibrary.org/obo/NCIT_C42781	http://purl.obolibrary.org/obo/NCIT_C42781
Study Person Roles Term Source REF	EFO				
