Historically, many 16S rRNA amplicon sequencing experiments were performed by using 454 (Roche) massively parallel pyrosequencing ( 10), both because it was the first commercially available system and because it later offered the longest read lengths, permitting interrogation of a larger and consequently more informative fraction of the 16S rRNA gene ( 11). General differences among next-generation sequencing platforms that may be used for this purpose, including relative turnaround times, per-base sequencing costs, read lengths, and accuracies, have been discussed elsewhere ( 7, – 9). For these reasons, 16S rRNA amplicon sequencing has found application in a wide range of metagenomic profiling studies, especially in those studies where speed or limited input material is a concern. As opposed to shotgun sequencing of genomic DNA extracted from a sample ( 4), wherein random fragments of bacterial genomes (and, potentially, contaminating DNA from host species or other organisms present) are sequenced and classified, 16S rRNA amplicon sequencing can be targeted specifically against bacteria, does not require the availability of reference genome sequences, and can be employed in cases where only trace amounts or poor-quality bacterial DNA templates are available ( 5, 6). One common approach for classifying microbial species present in a sample, rooted in previous cloning-based methods ( 3), is to PCR amplify a fraction of the taxonomically informative 16S rRNA gene and subject this product to next-generation DNA sequencing, enabling the classification of individual reads to specific taxa.
There has been increasing interest in exploring the bacterial communities that populate environmental ( 1) and biological ( 2) specimens. This study demonstrates the potential for differential bias in bacterial community profiles resulting from the choice of sequencing platform alone.
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Disparities could be attributed to the failure to generate full-length reads for particular organisms on the Ion Torrent platform, organism-dependent differences in sequence error rates affecting classification of certain species, or some combination of these factors. Results of bacterial community profiling performed on the mock community and a collection of 18 human-derived microbiological specimens were generally in good agreement for both platforms however, in some cases, results differed significantly. We found that these sequencing artifacts could be minimized by using bidirectional amplicon sequencing and an optimized flow order on the Ion Torrent platform. Read truncation was dependent on both the directionality of sequencing and the target species, resulting in organism-specific biases in community profiles. We observed comparatively higher error rates with the Ion Torrent platform and report a pattern of premature sequence truncation specific to semiconductor sequencing. We benchmarked performance by using a 20-organism mock bacterial community and a collection of primary human specimens. Here we compare the performances of two common “benchtop” sequencing platforms, Illumina MiSeq and Ion Torrent Personal Genome Machine (PGM), for bacterial community profiling by 16S rRNA (V1-V2) amplicon sequencing. High-throughput sequencing of the taxonomically informative 16S rRNA gene provides a powerful approach for exploring microbial diversity.