High quality single amplicon sequencing method for illumina Miseq platforms using pool of N spacer-linked target specific primers without PhiX spike-in

Illumina sequencing platform requires base diversity in the initial 11 cycles for efficient cluster identification and colour matrix estimation. This limitation yields low-quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but reduces the overall number of sequencing reads for data analysis. To overcome such low diversity issues during amplicon sequencing on Illumina platforms, we developed high throughput single amplicon sequencing method by introducing N spacers in target gene amplification primers that are pooled for simple handling. To demonstrate the precision of our method we performed a comparative study with standard illumina method using a commercial mock community DNA standard (ZymoBIOMICSTM). Towards this we performed two independent runs and the generated reads that were further analyzed using DADA2 pipeline.