INFLUENCES OF RANDOMNESS, AMPLIFICATION ERROR, AND BARCODE SEQUENCES ON MICROBIOTA STRUCTURE ANALYSIS THROUGH HIGH-THROUGHPUT SEQUENCING OF 16S rDNA AMPLICONS

High-throughput sequencing of 16S rDNA amplicons is widely used to analyse prokaryotic community structure. However, influence of the error introduced by PCR amplification process on the results is not well evaluated. To evaluate the influences of randomness, amplification error, and barcode sequences on microbiota structure analysis through high-throughput sequencing of 16S rDNA amplicons, we used primers with three different barcode sequences to sequence and compare the high-throughput sequencing results of 16S rDNA amplicons of Tegillarca granosa gut microbiota. Our results showed that removing chimeric sequences, different barcoding sequences, and different sequencing depths did not fundamentally change the differences in the microbiota structure and microbiota with different treatments were still aggregated according to the samples. Removing chimeric sequences and adopting different barcode sequences did not obviously affect the composition of the T. granosa gut microbiota at the phylum and genus levels. Chimeric sequences mainly led to overestimation of the alpha-diversity of the microbiota and the number of rare OTUs, whereas their impact on the number of rare OTUs was insufficient, and random factors had a higher impact than chimeric sequences. When analysing the beta-diversity of the microbiota and dominant OTUs, chimeric sequences had little impact on the results.