Impact of storage conditions on metabolite profiles of sputum samples from persons with cystic fibrosis  Jiangchao Zhao, Charles R. Evans, Lisa A. Carmody, John J. LiPuma  Journal of Cystic Fibrosis  Volume 14, Issue 4, Pages 468-473 (July 2015) DOI: 10.1016/j.jcf.2015.02.004 Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 1 Experimental design. The sample from the first individual (sputum sample 1) was stored at 4°C overnight before it was divided into 16 aliquots (50μL each). Two of the aliquots were stored at −80°C, while others were stored in duplicate at 4°C or −20°C for 1week, 2weeks, or 4weeks. Two aliquots were stored at room temperature for 1week. The sputum samples from the second and third individuals (sputum sample 2 and sputum sample 3) were each divided into 20 aliquots (50μL each) immediately after collection. Two aliquots of each sample were stored at −80°C immediately, while others were stored in duplicate at 4°C or −20°C for 1day, 5days, 12days or 28days. Two aliquots of each sample were stored at room temperature for 5days. After the specified storage period, all aliquots were stored at −80°C. Journal of Cystic Fibrosis 2015 14, 468-473DOI: (10.1016/j.jcf.2015.02.004) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 2 Effect of storage conditions on global metabolomic profiles in CF sputum. Principle coordinate analysis based on Bray–Curtis distances between metabolomic profiles of sputum sample aliquots is shown for sputum sample 1 (blue), sputum sample 2 (green), and sputum sample 3 (pink). Solid circles represent aliquots stored immediately at −80°C. Solid squares represent aliquots stored at room temperature for one week. Open circles and open triangles represent samples stored at −20°C and 4°C, respectively. For each of the three sputum samples, the sizes of the open symbols represent aliquots stored for increasing lengths of time as described in Fig. 1. Journal of Cystic Fibrosis 2015 14, 468-473DOI: (10.1016/j.jcf.2015.02.004) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 3 Effect of storage conditions on individual metabolites in CF sputum. Representative metabolites from sample 1 that showed a significant increase (A) or decrease (B) in concentration after storage at 4°C, compared to storage at −20°C. Results of replicate assays are depicted in each graph. Journal of Cystic Fibrosis 2015 14, 468-473DOI: (10.1016/j.jcf.2015.02.004) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions

Fig. 4 Microbiome profiles of 16S rDNA sequencing. The relative abundances of the bacterial taxa detected in each sample by next generation DNA sequencing are depicted. The identities of the five most abundant taxa are provided in the key. Journal of Cystic Fibrosis 2015 14, 468-473DOI: (10.1016/j.jcf.2015.02.004) Copyright © 2015 European Cystic Fibrosis Society. Terms and Conditions