This study presents a protocol for ultra-high-throughput microbial community analysis using the Illumina HiSeq2000 and MiSeq platforms. The protocol was applied to sequence 24 microbial communities from host-associated and free-living environments. The results show that biological conclusions derived from these platforms are consistent across different instruments and sequencing regions. The HiSeq2000 and MiSeq platforms differ in scale, with the HiSeq2000 producing significantly more data than the MiSeq. However, the results from both platforms are highly reproducible, as demonstrated by Procrustes analysis of principal coordinates plots. The study also shows that the cost of sequencing continues to decrease, making high-throughput microbial ecology more accessible. The protocol is suitable for large-scale microbial community analysis, such as the Earth Microbiome Project. The study highlights the importance of using multiple samples rather than increasing sequencing depth for beta diversity analysis, as deeper sequencing does not necessarily provide additional insights. The findings suggest that the decreased cost of sequencing should be used to study microbial systems with more samples rather than more sequences per sample. The study also notes that while a small number of sequences per sample may be sufficient for differentiating environments, more sequences are needed for similar environments. The study concludes that the protocol is ready for widespread use in microbial community analysis. The authors disclose that several authors are employees of Illumina, Inc. The study was funded by various organizations, including Amazon Web Services, NIH, and the Howard Hughes Medical Institute.This study presents a protocol for ultra-high-throughput microbial community analysis using the Illumina HiSeq2000 and MiSeq platforms. The protocol was applied to sequence 24 microbial communities from host-associated and free-living environments. The results show that biological conclusions derived from these platforms are consistent across different instruments and sequencing regions. The HiSeq2000 and MiSeq platforms differ in scale, with the HiSeq2000 producing significantly more data than the MiSeq. However, the results from both platforms are highly reproducible, as demonstrated by Procrustes analysis of principal coordinates plots. The study also shows that the cost of sequencing continues to decrease, making high-throughput microbial ecology more accessible. The protocol is suitable for large-scale microbial community analysis, such as the Earth Microbiome Project. The study highlights the importance of using multiple samples rather than increasing sequencing depth for beta diversity analysis, as deeper sequencing does not necessarily provide additional insights. The findings suggest that the decreased cost of sequencing should be used to study microbial systems with more samples rather than more sequences per sample. The study also notes that while a small number of sequences per sample may be sufficient for differentiating environments, more sequences are needed for similar environments. The study concludes that the protocol is ready for widespread use in microbial community analysis. The authors disclose that several authors are employees of Illumina, Inc. The study was funded by various organizations, including Amazon Web Services, NIH, and the Howard Hughes Medical Institute.