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 a high throughput single amplicon sequencing method by introducing 'N' (0-10) spacers in target gene amplification primers that are pooled for simple handling.
We evaluated the efficiency of 'N' (0-10) spacer-linked primers by targeting bacterial 16S V3-V4 region, demonstrating heterogeneous base library construction. The addition of 'N' (0-10) spacers causes sequencing frameshift at every base that leads to base diversity and produces heterogeneous high quality reads within a single amplicon library. We have written a python based command-line software,"MetReTrim", to trim the 'N' (0-10) spacers from the raw reads ( https://github.com/Mohak91/MetReTrim ). We further demonstrated the accuracy of this method by comparative mock community analysis with standard illumina V3-V4 primer method. The ZymoBIOMICS™ microbial community DNA standard was used as a control for this study. We performed data analysisusing the DADA2 pipeline where taxonomy was assigned using SILVA database as reference. We ob...

High-quality single amplicon sequencing method for illumina MiSeq platform using pool of ‘N’ (0–10) spacer-linked target specific primers without PhiX spike-in

10.1186/s12864-023-09233-4