16s_V3-V4 region Sequencing of mock sample with N spacer-linked 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.