This review discusses the tools, techniques, and challenges in microbial community profiling for human microbiome projects. It highlights the strengths and weaknesses of various experimental approaches, sequencing methodologies, and analytical methods. A key question raised is whether there is a substantial core of abundant organisms or lineages shared by all individuals. The review suggests that in some habitats, such as the gut, diversity among individuals is so great that no species is consistently abundant across all individuals. Instead, the focus should be on higher-level taxa or functional genes. The human microbiota consists of about 100 trillion microbial cells, outnumbering human cells 10 to 1. These microbes provide a wide range of metabolic functions. Characterizing the microbiome is essential for completing the human genome. Two main methods for this characterization are small-subunit ribosomal RNA (rRNA) studies and metagenomic studies. rRNA studies use stable phylogenetic markers to define microbial lineages, while metagenomic studies analyze community DNA through shotgun sequencing. Techniques like metatranscriptomics and metaproteomics are useful for simpler microbial communities but are just beginning to be applied to human-associated communities. High-throughput sequencing has enabled significant progress in characterizing the human microbiome and its role in health and disease. These studies are challenging due to the scale and complexity of the microbiome and the variability between individuals. The review covers experimental and analytical techniques used to characterize human and mammalian microbiomes. It discusses the potential for a core human microbiome and the implications for microbiome studies. The review also addresses key questions such as whether changes in microbial abundance are generally important and how to perform sequencing. It discusses the choice of sequencing technology, read length, sampling depth, and the region of the rRNA to sequence. It emphasizes the importance of choosing the right region and primers to minimize bias and ensure accurate results. The review highlights the trade-offs between depth of coverage and the number of samples, and the importance of standardizing methods for reliable comparisons. The review concludes that while there is no single "best" region for rRNA sequencing, certain regions like V2 and V4 are recommended for their accuracy in taxonomy assignment. It also discusses the importance of using barcoded pyrosequencing to tag samples and avoid pooling, which can introduce variability. The review emphasizes the need for careful data analysis, including filtering low-quality reads and distinguishing between taxon-based and phylogenetic analyses. Both approaches provide valuable insights, and the choice between them depends on the study's goals. The review underscores the importance of understanding the microbiome's complexity and variability to advance our knowledge of its role in health and disease.This review discusses the tools, techniques, and challenges in microbial community profiling for human microbiome projects. It highlights the strengths and weaknesses of various experimental approaches, sequencing methodologies, and analytical methods. A key question raised is whether there is a substantial core of abundant organisms or lineages shared by all individuals. The review suggests that in some habitats, such as the gut, diversity among individuals is so great that no species is consistently abundant across all individuals. Instead, the focus should be on higher-level taxa or functional genes. The human microbiota consists of about 100 trillion microbial cells, outnumbering human cells 10 to 1. These microbes provide a wide range of metabolic functions. Characterizing the microbiome is essential for completing the human genome. Two main methods for this characterization are small-subunit ribosomal RNA (rRNA) studies and metagenomic studies. rRNA studies use stable phylogenetic markers to define microbial lineages, while metagenomic studies analyze community DNA through shotgun sequencing. Techniques like metatranscriptomics and metaproteomics are useful for simpler microbial communities but are just beginning to be applied to human-associated communities. High-throughput sequencing has enabled significant progress in characterizing the human microbiome and its role in health and disease. These studies are challenging due to the scale and complexity of the microbiome and the variability between individuals. The review covers experimental and analytical techniques used to characterize human and mammalian microbiomes. It discusses the potential for a core human microbiome and the implications for microbiome studies. The review also addresses key questions such as whether changes in microbial abundance are generally important and how to perform sequencing. It discusses the choice of sequencing technology, read length, sampling depth, and the region of the rRNA to sequence. It emphasizes the importance of choosing the right region and primers to minimize bias and ensure accurate results. The review highlights the trade-offs between depth of coverage and the number of samples, and the importance of standardizing methods for reliable comparisons. The review concludes that while there is no single "best" region for rRNA sequencing, certain regions like V2 and V4 are recommended for their accuracy in taxonomy assignment. It also discusses the importance of using barcoded pyrosequencing to tag samples and avoid pooling, which can introduce variability. The review emphasizes the need for careful data analysis, including filtering low-quality reads and distinguishing between taxon-based and phylogenetic analyses. Both approaches provide valuable insights, and the choice between them depends on the study's goals. The review underscores the importance of understanding the microbiome's complexity and variability to advance our knowledge of its role in health and disease.