Independent Validation of Differential Abundance Patterns from Illumina Miseq Analysis Using Quantitative PCR Techniques on the Selective Primer for Chitinophaga

A criticism of amplicon sequencing is the potential for bias during PCR amplification. Quantitative PCR (qPCR) is an independent validation that can estimate taxon abundance and confirm patterns observed in amplicon sequencing patterns. Therefore, the objective was to design primers based on NGS sequencing and test qPCR primers to validate abundance patterns of bacterial and fungal OTUs on soils from the Optimized Shrub-intercropping System (OSS) or Sole Cropping in the Sahel. The results showed that quantitative PCR (qPCR) independently validated patterns observed in high throughput sequencing (HTS) analyses. Specific sub-genus level OTU clusters were found to be significantly enriched in intercropped millet plants in an experiment using the Ilumina MiSeq platform. These OTU sequences were used to design primers to independently validate the trends observed in that study. A total of seven OTU clusters were targeted in the Aspergillus, Chitinophaga, Fusarium, Lasiodiplodia, and Penicillium genera. The majority of those primers showed poor specificity for their intended targets, while the Chitinophaga specific primer set showed clear amplification with a single band at the expected size. This primer was used for qPCR analysis of the same DNA templates used for the Illumina MiSeq study. Quantitative PCR shows significant (P < 0.05) enrichment of Chitinophaga marker DNA that match the previously observed patterns. MiSeq analysis showed two times higher fold change differences in markers than observed in the qPCR study. These results demonstrate that selective primers can be designed from OTU sequence data and that qPCR analysis can be utilized to independently validate trends observed in HTS studies.