{"status":"ok","message-type":"work","message-version":"1.0.0","message":{"indexed":{"date-parts":[[2026,10,30]],"date-time":"2026-10-30T02:21:47Z","timestamp":1793326907768,"version":"4.5.0"},"reference-count":173,"publisher":"MDPI AG","issue":"2","license":[{"start":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T00:00:00Z","timestamp":1770336000000},"content-version":"vor","delay-in-days":0,"URL":"https:\/\/creativecommons.org\/licenses\/by\/4.0\/"}],"content-domain":{"domain":[],"crossmark-restriction":false},"short-container-title":["Microorganisms"],"abstract":"<jats:p>The human microbiome is increasingly recognized as a key determinant of health and disease, yet methodological variability continues to limit reproducibility and clinical translation of findings. This review synthesizes current approaches in microbiome research, critically evaluating each step from sampling to sequencing and downstream bioinformatics. Pre-analytical factors such as sample type, collection method, preservation, and storage conditions profoundly affect microbial community profiles and remain a major source of bias. Nucleic acid extraction protocols and quality assessment strategies are discussed with emphasis on optimized lysis techniques, contamination controls, and DNA yield evaluation. Advances in sequencing technologies are highlighted, including 16S rRNA amplicon sequencing, shotgun metagenomics, third-generation long-read platforms, and emerging single-cell and minimal-input methods, each with specific advantages and limitations in taxonomic and functional resolution. Bioinformatics pipelines for taxonomic profiling, variant detection, phylogenetic inference, and functional annotation are compared, with attention to widely used reference databases such as RefSeq, GTDB, and SILVA. Integrative multi-omics approaches, including metatranscriptomics, metabolomics, and genome-scale metabolic modeling, are presented as powerful tools for linking microbial community structure to host physiology and disease mechanisms. Despite these advances, the lack of standardized workflows across pre-analytical, sequencing, and computational steps continues to hinder inter-study comparability and biomarker validation. This review aims to provide a methodological framework that highlights both strengths and limitations of current technologies while underlining the need for harmonized protocols to ensure reproducibility and accelerate the translation of microbiome research into clinical practice.<\/jats:p>","DOI":"10.3390\/microorganisms14020387","type":"journal-article","created":{"date-parts":[[2026,2,6]],"date-time":"2026-02-06T10:05:47Z","timestamp":1770372347000},"page":"387","update-policy":"https:\/\/doi.org\/10.3390\/mdpi_crossmark_policy","source":"Crossref","is-referenced-by-count":18,"title":["Basic Microbiome Analysis: Analytical Steps from Sampling to Sequencing"],"prefix":"10.3390","volume":"14","author":[{"ORCID":"https:\/\/orcid.org\/0000-0003-4869-8199","authenticated-orcid":false,"given":"G\u00fclfem","family":"Ece","sequence":"first","affiliation":[{"name":"Department of Medical Microbiology, \u0130zmir City Hospital, \u0130zmir 35540, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-3243-6338","authenticated-orcid":false,"given":"Ahmet","family":"Akta\u015f","sequence":"additional","affiliation":[{"name":"\u0130stanbul Public Health Laboratory No. 2, \u0130stanbul Provincial Health Directorate, \u0130stanbul 34524, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-1260-0671","authenticated-orcid":false,"given":"\u00d6zlem","family":"Koyuncu \u00d6zyurt","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Akdeniz Univertsity, Antalya 07070, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-4180-8201","authenticated-orcid":false,"given":"Hadiye","family":"Demirbakan","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Sanko University, Gaziantep 27090, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0001-9060-3195","authenticated-orcid":false,"given":"Hikmet Eda","family":"Al\u0131\u015fkan","sequence":"additional","affiliation":[{"name":"Department of Microbiology, Adana Faculty of Medicine, Health Science University, Adana 01790, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0864-4989","authenticated-orcid":false,"given":"\u0130mran","family":"Sa\u011fl\u0131k","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Uludag University, Bursa 16059, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9645-7085","authenticated-orcid":false,"given":"Or\u00e7un","family":"Zorbozan","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, 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Medicine, Ankara University, Ankara 06230, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-9523-0380","authenticated-orcid":false,"given":"Emel","family":"Uzuno\u011flu","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Giresun University, Giresun 28200, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-0732-6471","authenticated-orcid":false,"given":"Esra","family":"Kaya","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Kahramanmara\u015f Necip Faz\u0131l City Hospital, Kahramanmara\u015f 46100, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-2747-0208","authenticated-orcid":false,"given":"Fatma","family":"Mutlu Sar\u0131g\u00fczel","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, 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T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0411-9669","authenticated-orcid":false,"given":"Mustafa","family":"Alt\u0131ndi\u015f","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Sakarya University, Sakarya 54290, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0003-0877-9162","authenticated-orcid":false,"given":"Sevin\u00e7","family":"Yenice Akta\u015f","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Onsekiz Mart University, \u00c7anakkale 17020, T\u00fcrkiye"}],"role":[{"vocabulary":"crossref","role":"author"}]},{"ORCID":"https:\/\/orcid.org\/0000-0002-2433-1977","authenticated-orcid":false,"given":"Tuba","family":"Kula Atik","sequence":"additional","affiliation":[{"name":"Department of Medical Microbiology, Faculty of Medicine, Bal\u0131kesir 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