High quality single amplicon sequencing method for illumina platforms using ‘N’ (0-10) spacer primer pool without PhiX spik-in

Illumina sequencing platform requires base diversity in initial 11 cycles for efficient cluster identification and color matrix estimation. This limitation yields low quality data for amplicon libraries having homogeneous base composition. Spike-in of PhiX library ensures base diversity but overall reduces the 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. We evaluated the efficiency of ‘N’ spacer primers by targeting bacterial 16S V3-V4 region, demonstrating heterogonous base library construction. Addition of ‘N’ spacer causes sequencing frame shift at every base that leads to base diversity and produces heterogenous high quality reads within single amplicon library. We have written a python script “MetReTrim” to trim the heterogenous ‘N’ spacers from the pre-processed reads. This method terminates the need for PhiX spike-in and allows for multiplexing of multiple samples, greatly reducing the overall cost and yields improved sequence quality.