{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,8,23]],"date-time":"2026-08-23T18:14:37Z","timestamp":1787508877523,"version":"build-2736575974"},"reference-count":38,"publisher":"Springer Science and Business Media LLC","issue":"1","license":[{"start":{"date-parts":[[2020,10,15]],"date-time":"2020-10-15T00:00:00Z","timestamp":1602720000000},"content-version":"tdm","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"},{"start":{"date-parts":[[2020,10,15]],"date-time":"2020-10-15T00:00:00Z","timestamp":1602720000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0"}],"funder":[{"DOI":"10.13039\/501100002850","name":"Fondo Nacional de Desarrollo Cient\u00edfico y Tecnol\u00f3gico,Chile","doi-asserted-by":"crossref","award":["1191311"],"award-info":[{"award-number":["1191311"]}],"id":[{"id":"10.13039\/501100002850","id-type":"DOI","asserted-by":"crossref"}]},{"name":"ERANet LAC","award":["ELAC2014\/HID-0254"],"award-info":[{"award-number":["ELAC2014\/HID-0254"]}]},{"DOI":"10.13039\/501100002850","name":"Fondo Nacional de Desarrollo Cient\u00edfico y Tecnol\u00f3gico","doi-asserted-by":"publisher","award":["1140412"],"award-info":[{"award-number":["1140412"]}],"id":[{"id":"10.13039\/501100002850","id-type":"DOI","asserted-by":"publisher"}]},{"name":"Spanish Ministry of Economy and Competitiveness","award":["SAF2012-31187"],"award-info":[{"award-number":["SAF2012-31187"]}]},{"DOI":"10.13039\/501100004587","name":"Instituto de Salud Carlos III","doi-asserted-by":"publisher","award":["ELAC2014\/HID-0254"],"award-info":[{"award-number":["ELAC2014\/HID-0254"]}],"id":[{"id":"10.13039\/501100004587","id-type":"DOI","asserted-by":"publisher"}]},{"DOI":"10.13039\/501100002704","name":"Fundaci\u00f3n Cient\u00edfica Asociaci\u00f3n Espa\u00f1ola Contra el C\u00e1ncer","doi-asserted-by":"publisher","award":["AECC 2017-1485"],"award-info":[{"award-number":["AECC 2017-1485"]}],"id":[{"id":"10.13039\/501100002704","id-type":"DOI","asserted-by":"publisher"}]}],"content-domain":{"domain":["link.springer.com"],"crossmark-restriction":false},"short-container-title":["Sci Rep"],"abstract":"<jats:title>Abstract<\/jats:title>\n                  <jats:p>Human lungs harbor a scarce microbial community, requiring to develop methods to enhance the recovery of nucleic acids from bacteria and fungi, leading to a more efficient analysis of the lung tissue microbiota. Here we describe five extraction protocols including pre-treatment, bead-beating and\/or Phenol:Chloroform:Isoamyl alcohol steps, applied to lung tissue samples from autopsied individuals. The resulting total DNA yield and quality, bacterial and fungal DNA amount and the microbial community structure were analyzed by qPCR and Illumina sequencing of bacterial 16S rRNA and fungal ITS genes. Bioinformatic modeling revealed that a large part of microbiome from lung tissue is composed of microbial contaminants, although our controls clustered separately from biological samples. After removal of contaminant sequences, the effects of extraction protocols on the microbiota were assessed. The major differences among samples could be attributed to inter-individual variations rather than DNA extraction protocols. However, inclusion of the bead-beater and Phenol:Chloroform:Isoamyl alcohol steps resulted in changes in the relative abundance of some bacterial\/fungal taxa. Furthermore, inclusion of a pre-treatment step increased microbial DNA concentration but not diversity and it may contribute to eliminate DNA fragments from dead microorganisms in lung tissue samples, making the microbial profile closer to the actual one.<\/jats:p>","DOI":"10.1038\/s41598-020-74137-2","type":"journal-article","created":{"date-parts":[[2020,10,15]],"date-time":"2020-10-15T06:04:27Z","timestamp":1602741867000},"update-policy":"https:\/\/doi.org\/10.1007\/springer_crossmark_policy","source":"Crossref","is-referenced-by-count":30,"title":["Optimized DNA extraction and purification method for characterization of bacterial and fungal communities in lung tissue samples"],"prefix":"10.1038","volume":"10","author":[{"given":"Vicente","family":"P\u00e9rez-Brocal","sequence":"first","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Fabien","family":"Magne","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Susana","family":"Ruiz-Ruiz","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Carolina A.","family":"Ponce","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Rebeca","family":"Bustamante","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Viviana San","family":"Martin","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Mireya","family":"Gutierrez","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Gianna","family":"Gatti","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Sergio L.","family":"Vargas","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]},{"given":"Andr\u00e9s","family":"Moya","sequence":"additional","affiliation":[],"role":[{"vocabulary":"crossref","role":"author"}]}],"member":"297","published-online":{"date-parts":[[2020,10,15]]},"reference":[{"key":"74137_CR1","doi-asserted-by":"publisher","first-page":"632","DOI":"10.1378\/chest.12-2854","volume":"144","author":"BJ Marsland","year":"2013","unstructured":"Marsland, B. J., Yadava, K. & Nicod, L. P. The airway microbiome and disease. Chest 144, 632\u2013637 (2013).","journal-title":"Chest"},{"key":"74137_CR2","doi-asserted-by":"publisher","first-page":"e88982","DOI":"10.1371\/journal.pone.0088982","volume":"9","author":"NA Kennedy","year":"2014","unstructured":"Kennedy, N. A. et al. The impact of different DNA extraction kits and laboratories upon the assessment of human gut microbiota composition by 16S rRNA gene sequencing. PLoS ONE 9, e88982 (2014).","journal-title":"PLoS ONE"},{"key":"74137_CR3","doi-asserted-by":"publisher","first-page":"130","DOI":"10.3389\/fmicb.2015.00130","volume":"6","author":"B Wagner Mackenzie","year":"2015","unstructured":"Wagner Mackenzie, B., Waite, D. W. & Taylor, M. W. Evaluating variation in human gut microbiota profiles due to DNA extraction method and inter-subject differences. Front. Microbiol. 6, 130 (2015).","journal-title":"Front. Microbiol."},{"key":"74137_CR4","doi-asserted-by":"publisher","first-page":"151","DOI":"10.1016\/j.syapm.2017.11.008","volume":"41","author":"MY Lim","year":"2018","unstructured":"Lim, M. Y., Song, E.-J., Kim, S. H., Lee, J. & Nam, Y.-D. Comparison of DNA extraction methods for human gut microbial community profiling. Syst. Appl. Microbiol. 41, 151\u2013157 (2018).","journal-title":"Syst. Appl. Microbiol."},{"key":"74137_CR5","doi-asserted-by":"publisher","first-page":"235","DOI":"10.1186\/s12879-016-1568-1","volume":"16","author":"NT Trung","year":"2016","unstructured":"Trung, N. T. et al. Enrichment of bacterial DNA for the diagnosis of blood stream infections. BMC Infect. Dis. 16, 235 (2016).","journal-title":"BMC Infect. Dis."},{"key":"74137_CR6","doi-asserted-by":"publisher","first-page":"227","DOI":"10.3390\/genes1020227","volume":"1","author":"M Knapp","year":"2010","unstructured":"Knapp, M. & Hofreiter, M. Next generation sequencing of ancient DNA: Requirements, strategies and perspectives. Genes (Basel) 1, 227\u2013243 (2010).","journal-title":"Genes (Basel)"},{"key":"74137_CR7","doi-asserted-by":"publisher","first-page":"4","DOI":"10.1016\/j.biocon.2014.11.019","volume":"183","author":"PF Thomsen","year":"2015","unstructured":"Thomsen, P. F. & Willerslev, E. Environmental DNA\u2014An emerging tool in conservation for monitoring past and present biodiversity. Biol. Conserv. 183, 4\u201318 (2015).","journal-title":"Biol. Conserv."},{"key":"74137_CR8","doi-asserted-by":"publisher","first-page":"89","DOI":"10.1016\/j.fsigen.2009.06.008","volume":"4","author":"LA Gefrides","year":"2010","unstructured":"Gefrides, L. A., Powell, M. C., Donley, M. A. & Kahn, R. UV irradiation and autoclave treatment for elimination of contaminating DNA from laboratory consumables. Forensic Sci. Int. Genet. 4, 89\u201394 (2010).","journal-title":"Forensic Sci. Int. Genet."},{"key":"74137_CR9","doi-asserted-by":"publisher","first-page":"789","DOI":"10.1016\/j.cell.2014.09.053","volume":"159","author":"JK Goodrich","year":"2014","unstructured":"Goodrich, J. K. et al. Human genetics shape the gut microbiome. Cell 159, 789\u2013799 (2014).","journal-title":"Cell"},{"key":"74137_CR10","doi-asserted-by":"publisher","first-page":"87","DOI":"10.1186\/s12915-014-0087-z","volume":"12","author":"SJ Salter","year":"2014","unstructured":"Salter, S. J. et al. Reagent and laboratory contamination can critically impact sequence-based microbiome analyses. BMC Biol. 12, 87 (2014).","journal-title":"BMC Biol."},{"key":"74137_CR11","doi-asserted-by":"publisher","first-page":"285","DOI":"10.1016\/j.mimet.2004.11.018","volume":"61","author":"T Mohammadi","year":"2005","unstructured":"Mohammadi, T., Reesink, H. W., Vandenbroucke-Grauls, C. M. J. E. & Savelkoul, P. H. M. Removal of contaminating DNA from commercial nucleic acid extraction kit reagents. J. Microbiol. Methods 61, 285\u2013288 (2005).","journal-title":"J. Microbiol. Methods"},{"key":"74137_CR12","doi-asserted-by":"publisher","first-page":"517","DOI":"10.1056\/NEJMoa1211115","volume":"369","author":"AS Bhatt","year":"2013","unstructured":"Bhatt, A. S. et al. Sequence-based discovery of Bradyrhizobium enterica in cord colitis syndrome. N. Engl. J. Med. 369, 517\u2013528 (2013).","journal-title":"N. Engl. J. Med."},{"key":"74137_CR13","doi-asserted-by":"publisher","first-page":"e83744","DOI":"10.1371\/journal.pone.0083744","volume":"9","author":"C Xuan","year":"2014","unstructured":"Xuan, C. et al. Microbial dysbiosis is associated with human breast cancer. PLoS ONE 9, e83744 (2014).","journal-title":"PLoS ONE"},{"key":"74137_CR14","doi-asserted-by":"publisher","first-page":"e8578","DOI":"10.1371\/journal.pone.0008578","volume":"5","author":"M Hilty","year":"2010","unstructured":"Hilty, M. et al. Disordered microbial communities in asthmatic airways. PLoS ONE 5, e8578 (2010).","journal-title":"PLoS ONE"},{"key":"74137_CR15","doi-asserted-by":"publisher","first-page":"957","DOI":"10.1164\/rccm.201104-0655OC","volume":"184","author":"ES Charlson","year":"2011","unstructured":"Charlson, E. S. et al. Topographical continuity of bacterial populations in the healthy human respiratory tract. Am. J. Respir. Crit. Care Med. 184, 957\u2013963 (2011).","journal-title":"Am. J. Respir. Crit. Care Med."},{"key":"74137_CR16","doi-asserted-by":"publisher","DOI":"10.1073\/pnas.1107435109","author":"AF Goddard","year":"2012","unstructured":"Goddard, A. F. et al. Direct sampling of cystic fibrosis lungs indicates that DNA-based analyses of upper-airway specimens can misrepresent lung microbiota. Proc. Natl. Acad. Sci. https:\/\/doi.org\/10.1073\/pnas.1107435109 (2012).","journal-title":"Proc. Natl. Acad. Sci."},{"key":"74137_CR17","doi-asserted-by":"publisher","first-page":"5724","DOI":"10.1038\/s41598-018-23989-w","volume":"8","author":"JL Pechal","year":"2018","unstructured":"Pechal, J. L., Schmidt, C. J., Jordan, H. R. & Benbow, M. E. A large-scale survey of the postmortem human microbiome, and its potential to provide insight into the living health condition. Sci. Rep. 8, 5724 (2018).","journal-title":"Sci. Rep."},{"key":"74137_CR18","doi-asserted-by":"publisher","first-page":"5435","DOI":"10.1038\/s41598-017-05882-0","volume":"7","author":"Y Zhu","year":"2017","unstructured":"Zhu, Y., Wang, L., Yin, Y. & Yang, E. Systematic analysis of gene expression patterns associated with postmortem interval in human tissues. Sci. Rep. 7, 5435 (2017).","journal-title":"Sci. Rep."},{"key":"74137_CR19","doi-asserted-by":"publisher","first-page":"170","DOI":"10.1097\/00019606-200209000-00008","volume":"11","author":"DP Me","year":"2002","unstructured":"Me, D. P. et al. Postmortem RNA and protein stability in perinatal human lungs. Diagn. Mol. Pathol. 11, 170\u2013176 (2002).","journal-title":"Diagn. Mol. Pathol."},{"key":"74137_CR20","doi-asserted-by":"publisher","first-page":"334","DOI":"10.1080\/21505594.2016.1235671","volume":"8","author":"L Tipton","year":"2017","unstructured":"Tipton, L., Ghedin, E. & Morris, A. The lung mycobiome in the next-generation sequencing era. Virulence 8, 334\u2013341 (2017).","journal-title":"Virulence"},{"key":"74137_CR21","doi-asserted-by":"publisher","first-page":"e357","DOI":"10.1016\/S1473-3099(17)30309-2","volume":"17","author":"DW Denning","year":"2017","unstructured":"Denning, D. W. & Chakrabarti, A. Pulmonary and sinus fungal diseases in non-immunocompromised patients. Lancet Infect. Dis. 17, e357\u2013e366 (2017).","journal-title":"Lancet Infect. Dis."},{"key":"74137_CR22","doi-asserted-by":"publisher","first-page":"1168","DOI":"10.3389\/fphys.2018.01168","volume":"9","author":"E Mathieu","year":"2018","unstructured":"Mathieu, E. et al. Paradigms of lung microbiota functions in health and disease, particularly, in asthma. Front. Physiol. 9, 1168 (2018).","journal-title":"Front. Physiol."},{"key":"74137_CR23","doi-asserted-by":"publisher","first-page":"29","DOI":"10.1186\/s40168-016-0172-3","volume":"4","author":"AP Lauder","year":"2016","unstructured":"Lauder, A. P. et al. Comparison of placenta samples with contamination controls does not provide evidence for a distinct placenta microbiota. Microbiome 4, 29 (2016).","journal-title":"Microbiome"},{"key":"74137_CR24","doi-asserted-by":"publisher","first-page":"405","DOI":"10.1078\/0723-2020-00060","volume":"24","author":"EG Zoetendal","year":"2001","unstructured":"Zoetendal, E. G., Ben-Amor, K., Akkermans, A. D., Abee, T. & de Vos, W. M. DNA isolation protocols affect the detection limit of PCR approaches of bacteria in samples from the human gastrointestinal tract. Syst. Appl. Microbiol. 24, 405\u2013410 (2001).","journal-title":"Syst. Appl. Microbiol."},{"key":"74137_CR25","doi-asserted-by":"publisher","first-page":"e33865","DOI":"10.1371\/journal.pone.0033865","volume":"7","author":"S Yuan","year":"2012","unstructured":"Yuan, S., Cohen, D. B., Ravel, J., Abdo, Z. & Forney, L. J. Evaluation of methods for the extraction and purification of DNA from the human microbiome. PLoS ONE 7, e33865 (2012).","journal-title":"PLoS ONE"},{"key":"74137_CR26","doi-asserted-by":"publisher","first-page":"5122","DOI":"10.1128\/JCM.43.10.5122-5128.2005","volume":"43","author":"DN Fredricks","year":"2005","unstructured":"Fredricks, D. N., Smith, C. & Meier, A. Comparison of six DNA extraction methods for recovery of fungal DNA as assessed by quantitative PCR. J. Clin. Microbiol. 43, 5122\u20135128 (2005).","journal-title":"J. Clin. Microbiol."},{"key":"74137_CR27","doi-asserted-by":"publisher","first-page":"e90899","DOI":"10.1371\/journal.pone.0090899","volume":"9","author":"AK Dupuy","year":"2014","unstructured":"Dupuy, A. K. et al. Redefining the human oral mycobiome with improved practices in amplicon-based taxonomy: Discovery of Malassezia as a prominent commensal. PLoS ONE 9, e90899 (2014).","journal-title":"PLoS ONE"},{"key":"74137_CR28","doi-asserted-by":"publisher","first-page":"2541e4","DOI":"10.1126\/scitranslmed.3009864","volume":"6","author":"HJ Kliman","year":"2014","unstructured":"Kliman, H. J. Comment on \u201cThe placenta harbors a unique microbiome\u201d. Sci. Transl. Med. 6, 2541e4 (2014).","journal-title":"Sci. Transl. Med."},{"key":"74137_CR29","doi-asserted-by":"publisher","first-page":"171","DOI":"10.1093\/cid\/cis870","volume":"56","author":"SL Vargas","year":"2013","unstructured":"Vargas, S. L. et al. Near-universal prevalence of Pneumocystis and associated increase in mucus in the lungs of infants with sudden unexpected death. Clin. Infect. Dis. 56, 171\u2013179 (2013).","journal-title":"Clin. Infect. Dis."},{"issue":"Suppl 1","key":"74137_CR30","doi-asserted-by":"publisher","first-page":"4516","DOI":"10.1073\/pnas.1000080107","volume":"108","author":"JG Caporaso","year":"2011","unstructured":"Caporaso, J. G. et al. Global patterns of 16S rRNA diversity at a depth of millions of sequences per sample. Proc. Natl. Acad. Sci. U. S. A. 108(Suppl 1), 4516\u20134522 (2011).","journal-title":"Proc. Natl. Acad. Sci. U. S. A."},{"key":"74137_CR31","doi-asserted-by":"publisher","first-page":"4356","DOI":"10.1128\/AEM.66.10.4356-4360.2000","volume":"66","author":"J Borneman","year":"2000","unstructured":"Borneman, J. & Hartin, R. J. PCR primers that amplify fungal rRNA genes from environmental samples. Appl. Environ. Microbiol. 66, 4356\u20134360 (2000).","journal-title":"Appl. Environ. Microbiol."},{"key":"74137_CR32","doi-asserted-by":"publisher","first-page":"581","DOI":"10.1038\/nmeth.3869","volume":"13","author":"BJ Callahan","year":"2016","unstructured":"Callahan, B. J. et al. DADA2: High-resolution sample inference from Illumina amplicon data. Nat. Methods 13, 581\u2013583 (2016).","journal-title":"Nat. Methods"},{"key":"74137_CR33","doi-asserted-by":"publisher","first-page":"357","DOI":"10.1038\/nmeth.1923","volume":"9","author":"B Langmead","year":"2012","unstructured":"Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357\u2013359 (2012).","journal-title":"Nat. Methods"},{"key":"74137_CR34","doi-asserted-by":"publisher","first-page":"335","DOI":"10.1038\/nmeth.f.303","volume":"7","author":"JG Caporaso","year":"2010","unstructured":"Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335\u2013336 (2010).","journal-title":"Nat. Methods"},{"key":"74137_CR35","unstructured":"R Core Team. R: A Language and Environment for Statistical Computing. (R Found Stat Comput Vienna, Austria, 2019)."},{"key":"74137_CR36","doi-asserted-by":"publisher","first-page":"290","DOI":"10.1890\/0012-9658(2001)082[0290:FMMTCD]2.0.CO;2","volume":"82","author":"BH McArdle","year":"2001","unstructured":"McArdle, B. H. & Anderson, M. J. Fitting multivariate models to community data: A comment on distance-based redundancy analysis. Ecology 82, 290\u2013297 (2001).","journal-title":"Ecology"},{"key":"74137_CR37","doi-asserted-by":"publisher","first-page":"550","DOI":"10.1186\/s13059-014-0550-8","volume":"15","author":"MI Love","year":"2014","unstructured":"Love, M. I., Huber, W. & Anders, S. Moderated estimation of fold change and dispersion for RNA-seq data with DESeq2. Genome Biol. 15, 550 (2014).","journal-title":"Genome Biol."},{"key":"74137_CR38","doi-asserted-by":"crossref","first-page":"e61217","DOI":"10.1371\/journal.pone.0061217","volume":"8","author":"PJ McMurdie","year":"2013","unstructured":"McMurdie, P. J. & Holmes, S. phyloseq: an R package for reproducible interactive analysis and graphics of microbiome census data. PLoS ONE 8, e61217 (2013).","journal-title":"PLoS ONE"}],"container-title":["Scientific Reports"],"original-title":[],"language":"en","link":[{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-74137-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-74137-2","content-type":"text\/html","content-version":"vor","intended-application":"text-mining"},{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-74137-2.pdf","content-type":"application\/pdf","content-version":"vor","intended-application":"similarity-checking"}],"deposited":{"date-parts":[[2022,12,6]],"date-time":"2022-12-06T16:33:17Z","timestamp":1670344397000},"score":1,"resource":{"primary":{"URL":"https:\/\/www.nature.com\/articles\/s41598-020-74137-2"}},"subtitle":[],"short-title":[],"issued":{"date-parts":[[2020,10,15]]},"references-count":38,"journal-issue":{"issue":"1","published-online":{"date-parts":[[2020,12]]}},"alternative-id":["74137"],"URL":"https:\/\/doi.org\/10.1038\/s41598-020-74137-2","relation":{},"ISSN":["2045-2322"],"issn-type":[{"value":"2045-2322","type":"electronic"}],"subject":[],"published":{"date-parts":[[2020,10,15]]},"assertion":[{"value":"19 March 2020","order":1,"name":"received","label":"Received","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"28 July 2020","order":2,"name":"accepted","label":"Accepted","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"15 October 2020","order":3,"name":"first_online","label":"First Online","group":{"name":"ArticleHistory","label":"Article History"}},{"value":"The authors declare no competing interests.","order":1,"name":"Ethics","group":{"name":"EthicsHeading","label":"Competing interests"}}],"article-number":"17377"}}