Changing of the Guard: Migrating 16S and ITS Diversity Profiling from 454 Onto Alternate NGS Platforms

摘要

The Australian Genome Research Facility (AGRF) currently operates a microbial diversity profiling service on the 454 GS-FLX platform, targeting various regions of 16S and ITS microbial genes. The recent announcement by Roche to discontinue support prompted us to investigate how this service can be transitioned to alternate NGS platforms. The objective of this study was to evaluate the accuracy and reproducibility of each platform/target combination for both artificial control samples (known gDNA pools) and natural microbial community samples (soils). Replicate PCR amplicons were generated for several microbial and fungal regions (16S and ITS), and sequenced on the GS-FLX, MiSeq and Ion Torrent PGM platforms. The resulting sequences were identified at different taxonomic levels (phylum to species) and the results for each sample, platform and target compiled. This comparison revealed distinct and reproducible differences between the same samples run on different platforms. While no platform was able to perfectly describe the proportions of the artificial pooled sample, each one showed a high level of reproducibility (< 1% variance). This suggests that PCR bias during amplification gives rise to consistent biases in resulting microbial distributions and confirms this method as a useful tool for comparing samples and monitoring changes, but demonstrate its weakness for absolute quantitation. Between platforms, the long reads of the GS-FLX gave the greatest resolution for microbial identification, followed by the MiSeq. Despite the shorter amplicon size, the ion torrent was still able to provide high resolution profiles of the microbial samples. These results show that currently, the 454 still provides higher resolution data sets for microbial diversity than other available platforms. However, as read lengths improve and error rates decrease, these alternate platforms should provide a suitable transition pathway for researchers interested in profiling microbial diversity.