Assessment of 16S rDNA hypervariable regions for screening bacterial diversity in complex soil environments with multimillion sequence read generating technologies

Multimillion read generating, high throughput sequencing technologies have become powerful with prospects in resolving 16S rDNA bacterial diversity of highly complex soil environments. However, contemporarily these technologies are length-wise restricted in screening DNA stretches of single 16S rDNA hypervariable (V) regions. Aim of the present study was to assess effects of properties of four consecutive V regions (V3-6) on commonly applied analytical methodologies in bacterial ecology studies. This was carried out by exploiting commonly used non-redundant 16S rDNA sequence databases and also simulated samples according to previous high throughput sequencing studies. Results indicate that primers targeting the mere 16S rDNA gene should be de novo designed for soil environments, since previous 16S rDNA conservation studies were based on databases heavily dominated by the less diverse human microbiome sequences with database participation of up to 54 %. V3 and V4 showed an overall higher representation of the full-length 16S rDNA variants with V4 however lacking highly conserved flanking sites that would allow primer designing for maximum diversity screening. V6 had the poorest performance throughout all analyses. Based on these results, theoretical assessment of the experimental approach is strongly suggested prior performance of 16S rDNA based bacterial diversity studies.