16s_V3-V4 region Sequencing of mock sample with standard Illumina method
To resolve the technical limitations of single amplicon sequencing on Illumina platforms and challenges encountered during heterogeneity spacer primer designing, we added N nucleotides to the 5' end of the gene-specific primers for amplifying the gene. The N nucleotide bases are added in 0-10 fashion in forward and reverse gene-specific primers.  A pool of N (0-10) spacers-linked gene-specific primers is used for amplification and library synthesis incorporating diversity within a single library.