New V4V6 approach

We have developed a new more flexible, precise and conceptually different approach for illumina 16S Microbiome analysis .

Using Optimal Primers, an Asymmetric Sequencing Design and a New Bioinformatics Algorithm of analysis especially designed for this approach, we are able to get high quality results and precise taxonomic assignments from non-contiguous regions sequencing.


Era7 V4V6 16S approach for bacterial taxonomic profiling


Description of our new V4V6 approach

Optimal primers

The V4V6 approach uses optimal primers located in 16S regions highly conserved in bacteria and archaea. The primers are not degenerated and have been designed to cover all the sequences included in 16SDB7, our database of 16S sequences (with more than 6 millions of sequences). Sets of degenerated primers can include primers with sequence combinations that are not in any target. It can interfere the PCR processes and even cause the amplification of undesired sequences.

Asymmetric sequencing design

In our V4V6 approach we sequence the complete V4 and V6 variable regions and their flanking sequences.  The sequencing design is asymmetric to better cover V4 and V6 regions using the selected optimal primers. R1 covers 180 bp and R2 120 bp.  Thus, using 300-cycle illumina reagents with runs of 180x120 we cover V4 and V6 complete regions.

Non-contiguous regions sequencing

Our new approach analyzes non-contiguous regions as a whole maintaining the maximal capacity of discrimination between sequences and, hence, getting the most specific taxonomic assignment for them. Another advantage over approaches based on overlapping reads is that we do not waste sequence in the overlapped region.

New bioinformatics algorithm of analysis especially designed for this approach

The bioinformatics analysis developed for this kind of approach is new and it is based on a conceptually different theoretical framework. The algorithm includes indexation of the references database using ultrametric spaces, a new modeling for DNA sequences and amplicons and new original computational strategies to optimize comparison time maintaining the maximal precision in the assignment.


For a detailed description of the new approach for 16S taxonomic profiling and of the new algorithm and mathematical modelling that we have developed for amplicon analysis see these Preprints:

  • Pareja-Tobes, E.; Tobes, R. Amplicon Analysis I. Preprints 2019, 2019090171 (doi: 10.20944/preprints201909.0171.v1).

  • Pareja-Tobes, E.; Tobes, R. ARSA-16S: A New and Conceptually Different Approach for 16S-based Taxonomic Profiling. Preprints 2019, 2019090170 (doi: 10.20944/preprints201909.0170.v1).