Comparing Apples and Oranges?: Next Generation Sequencing and Its Impact on Microbiome Analysis

• DOI: 10.1371/journal.pone.0148028
• Publication Date: 2016-02-05T18:55:55Z
• Authors: Adam G. Clooney; Fiona Fouhy; Roy D. Sleator; Aisling O’ Driscoll; Catherine Stanton; Paul D. Cotter; Marcus J. Claesson
• Keywords: Amplicon sequencing; Amplicon; Ion semiconductor sequencing; Massive parallel sequencing

Rapid advancements in sequencing technologies along with falling costs present widespread opportunities for microbiome studies across a vast and diverse array of environments. These impressive technological developments have been accompanied by considerable growth the number methodological variables, including sampling, storage, DNA extraction, primer pairs, technology, chemistry version, read length, insert size, analysis pipelines, amongst others. This increase variability threatens to compromise both reproducibility comparability conducted. Here we perform first reported study comparing amplicon shotgun three leading next-generation technologies. were applied six human stool samples using Illumina HiSeq, MiSeq Ion PGM sequencing, as well two variable 16S rRNA gene regions. Notably, found that factor responsible greatest variance microbiota composition was chosen methodology rather than natural inter-individual variance, which is commonly one most significant drivers studies. Amplicon suffered from this large extent, issue particularly apparent when V1-V2 region amplicons sequenced MiSeq. Somewhat surprisingly, choice taxonomic binning software sequences proved be crucial importance even greater discriminatory power technology amplicon. Optimal N50 assembly values HiSeq obtained 10 million reads per sample, whereas depths less sufficient samples. The latter technologies, on other hand, provide better basis functional categorisation, possibly due their longer lengths. Hence, addition highlighting biases, demonstrates risks associated data generated different strategies. We also recommend laboratories particular interests certain microbes should optimise protocols accurately detect these taxa techniques.