The Comparative Analysis And Discussion Of 16S RRNA Sequencing Data Based On 454 And Ion Torrent Platform

The sequence information of 16 S r RNA gene has been widely used in the study of species identification and phylogenetics at present. The 16 S r RNA gene has been sequenced by the sequencing platforms such as Illumina, 454, as well as the latest Ion Torrent PGM(Personal Genome Machine). The same sample obtained by different sequencing platforms resulted with different, how to reduce the bias arising from the sequencing platform so that the final result is more accurate and reliable has always been the concern of the majority of scientific researchers. We formed a 16 S r RNA analysis process based on the latest Ion Torrent PGM platform first of all.We collected feces samples of six mice fed for 8 weeks, using 454 pyrophosphate sequencer(454 Life Sciences, Roche, USA), Ion Torrent PGM sequencing respectively. In order to evaluate the stability of Ion Torrent PGM, the samples 0121100852 and 0121100859 were sequenced for three times by applying forward sequencing(V4-V5) and reverse sequencing(V5-V4), while the reverse sequencing(V5-V3) was applied for 454 platform. According to the data volume and OTU(Operational taxonomic units) distribution of the 26 sequenced samples, the reverse sequencing V54 reads number for each sample will be nearly saturated when it comes to 30000; Since Ion Torrent PGM sequencing V45 read length is shorter than the reverse sequencing, it will need more reads to get saturated, and we suggest about 40000 reads for each farward seqencing sample. The species annotation proportion of the samples sequenced by the Ion Torrent PGM farward sequencing is lower than the reverse at the class, order and family level, however, is higher than that at the genera and species level. The same samples that were repetitively sequenced show high consistency by the comparative analysis of the species annotation, the PCo A(principal coordinates analysis), Procrustes analysis and beta diversity comparation, and this illustrated the sequencing stability of Ion Torrent PGM platform is quite good. The base quality and read length of the 454 platform are better than the Ion Torrent PGM platform, while the Procrustes correlation of the profiles of Ion Torrent PGM and 454 reverse sequencing at the OTU, phylum, class, order, and family, genera and species level is high(0.783~0.9169), along with the PCo A(principal coordinates analysis) and the evolutionary tree drawed by beta diversity display the samples are clustered by the sample source, and the permanova(nonparametric multivariate analysis of variance) analysis discover the p value of the factor of sequencing platform is 0.235 while the factor of the sample source is 0.001. We can conclude from the above that the main variation is the sample source is for our 26 samples rather than the sequencing platform. Meanwhile, the species annotation proportion of the Ion Torrent PGM platform is slightly higher than the 454 platform at all the species levels(the species annotation ratio is a very important index for evaluating the sequencing data quality), which shows the Ion Torrent PGM platform has an advantage, moreover, considering the low cost, fast sequencing and other unique advantages of this new sequencing platform, we think that the Ion Torrent PGM platform can completely replace the 454 for the 16 S r RNA analysis.