# Heterogeneity Primer Spacers Improve the Performance of Massively Parallel Amplicon Sequencing of the V3-V4 Region of the 16 S rDNA as well as the 18 S Region for Blastocystis Subtyping

## TL;DR

This paper introduces a new method using heterogeneity spacers in PCR primers to improve sequencing performance for 16S and 18S rDNA marker genes.

## Contribution

A systematic, algorithm-driven approach to designing heterogeneity spacers that increase sequencing diversity and eliminate the need for PhiX balancing.

## Key findings

- Heterogeneity spacers improved sequencing signal diversity in 16S and 18S rDNA profiling.
- The method was successfully applied to thousands of samples without requiring PhiX library balancing.
- Staggered primer designs with spacers ensured equal representation of primer combinations.

## Abstract

The Illumina MiSeq and NextSeq instruments are workhorses for many low- to medium-throughput amplicon sequencing applications in microbiology; however, their performance is highly sensitive to decreased diversity of libraries. Here, we present a systematic, algorithm-driven approach to designing heterogeneity spacers for amplification primers and demonstrate their use in two marker gene profiling applications. Heterogeneity spacers were designed to balance the sequencing signal, ensuring that no sequencing cycle contained more than 60% of a single dominant base. Additional bases were inserted into the first-round amplification primer adjacent to the target-specific portion. A simple script was written to remove the heterogeneity spacers from the reads, and verify equal representation of primer combinations. We designed sixteen staggered primer variants for profiling the V3-V4 regions of the 16S bacterial rDNA gene and sixteen for bidirectional profiling of the 18S rDNA region informative for Blastocystis subtypes. The effectiveness of our design was evident from the increased heterogeneity of the sequencing signal, with no primer combination predominating. The method was tested against classical primer design and has successfully been applied to several thousand samples to date. Inserting heterogeneity spacers before the target-specific portion of the first-round PCR primers is a simple and reliable way to increase the diversity of marker gene amplicons. The multiplexed reactions produce amplicons that can be easily sequenced on Illumina platforms, eliminating the need for high concentrations of balancing PhiX library.

The online version contains supplementary material available at 10.1007/s00248-026-02708-3.