ena-STUDY-UCONNMISEQ-04-10-2013-16:36:50:396-217
Comparison of 454 and Illumina 16S sequencing
High throughput 16S sequencing surveys have become widely used to examine microbial communities in a broad range of environments. While initial studies used pyrosequencing platforms from Roche/454 Life Science, Illumina sequencing systems are becoming more widely adopted due to lower associated costs and higher data output. While initial studies examining the feasibility of Illumina based 16S sequencing have shown that beta diversity comparisons were comparable to those produced by pyrosequencing, it has yet to be demonstrated if pre-existing 454 pyrosequencing primers and analysis pipelines can be directly transferred from 454 to Illumina sequencing by changing sequencing adapters of the primers. In this study we modified pre-existing 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencing instruments. Six microbial communities were analyzed by 454 and Illumina sequencing using the V4-V5 primers, along with Illumina sequencing of the V4 region using published primers. The data was analyzed using both reference-based and de novo OTU clustering pipelines to determine biases unique to each method. Beta diversity analyses confirmed that both sequencing technologies provide similar results, however measures of alpha diversity and taxonomic composition yielded differences resulting from the choice of analysis pipeline. While moving to Illumina based sequencing promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.
High throughput 16S sequencing surveys have become widely used to examine microbial communities in a broad range of environments. While initial studies used pyrosequencing platforms from Roche/454 Life Science, Illumina sequencing systems are becoming more widely adopted due to lower associated costs and higher data output. While initial studies examining the feasibility of Illumina based 16S sequencing have shown that beta diversity comparisons were comparable to those produced by pyrosequencing, it has yet to be demonstrated if pre-existing 454 pyrosequencing primers and analysis pipelines can be directly transferred from 454 to Illumina sequencing by changing sequencing adapters of the primers. In this study we modified pre-existing 454 pyrosequencing primers targeting the V4-V5 hyper-variable regions of the 16S rRNA gene to be compatible with Illumina sequencing instruments. Six microbial communities were analyzed by 454 and Illumina sequencing using the V4-V5 primers, along with Illumina sequencing of the V4 region using published primers. The data was analyzed using both reference-based and de novo OTU clustering pipelines to determine biases unique to each method. Beta diversity analyses confirmed that both sequencing technologies provide similar results, however measures of alpha diversity and taxonomic composition yielded differences resulting from the choice of analysis pipeline. While moving to Illumina based sequencing promises to provide deeper insights into the breadth and function of microbial diversity, our results show that care must be taken to ensure that sequencing and processing artifacts do not obscure true microbial diversity.