Abstract
Culture-independent molecular techniques and advances in next generation sequencing (NGS) technologies make large scale epidemiological studies on microbiota feasible. A challenge using NGS is to obtain the sufficient sequence depth and accuracy to ensure high reproducibility and repeatability which is mostly attained through robust amplification. Here, we aimed to assess the reproducibility of saliva microbiota profiles produced with simplified in-house 16S amplicon assays with large number of barcodes by comparing triplicate samples. The assays included primers with Truseq (TS-tailed) or Nextera (NX-tailed) adapters and either with dual index or dual index plus a 6-bp internal index. All amplification protocol produced consistent microbial profiles for the same samples. However, in our study, reproducibility was highest for the TS-tailed method. Five replicates of a single sample, prepared with the TS-tailed 1-step protocol without internal index sequenced on the HiSeq platform provided high alpha-diversity and low standard deviation (mean Shannon and Inverse Simpson diversity was 3.19 ± 0.097 and 13.56 ± 1.634 respectively). This proposes that 16S amplicon assays using numerous barcodes suitable for large samples sizes and the TS-tailed protocol without internal index can be considered an accurate protocol that provides consistent quantification of bacterial profiles.