Abstract
Purpose :
Characterize the differences in conjunctival microbiome profiles based on two minor variations in a standard next generation sequencing (NGS) library preparation technique to facilitate production of 16S rRNA metagenomics data from extremely low yield specimens. Results were analyzed with three different 16S bioinformatics pipelines. In addition, DNA shotgun sequencing was also performed and results were compared with the 16S analysis.
Methods :
Seven subjects were enrolled. Conjunctival samples were taken bilaterally and combined into a single vial with DNA preservative for sequencing. After extraction, the DNA yield was insufficient for typical 16S rRNA NGS processing according to standard protocols. Given the low DNA yield, the 16S rRNA library preparation was altered to enable analysis of the specimens. Two separate methods were tried, and both produced metagenomics profiles for all of the samples. One involved following the library prep protocol as directed with the modification of loading as much sample possible even when no DNA was detected. The second involved an additional round of index PCR with the products from the first round utilized in the second so that sufficient DNA for preparation of the NGS library for sequencing was produced.
Results :
The additional round of PCR produced an average of over 500,000 reads per sample but required removal of contaminant taxa based on results from the negative control. Without the additional round of PCR contaminant taxa was far less prevalent in the negative control but there was only an average of a little over 12,000 reads per sample. Both 16S methods and all three bioinformatics pipelines utilized produced similar results. Shotgun sequencing results correlated to the extent expected with the 16S results.
Conclusions :
There was very little variability between the different 16S sequencing techniques in regards to the most common bacteria. Even though there were only subtle differences in the 16S metagenomics profiles of the specimens, additional studies are needed before definitive recommendations regarding the use of a second round of PCR to increase read or utilizing the costlier shotgun sequencing approach provides an optimal assessment of the ocular surface microbiome.
This is a 2021 ARVO Annual Meeting abstract.