Environmental RNA metabarcoding for ballast water microbial diversity: Minimizing false positives

While maritime transport boosts global trade by shipping bulk goods, it raises concerns about the spread of harmful bacteria via ballast water. Moreover, the dark and cold environments of ballast tanks often harbor extracellular DNA from dead organisms, leading to false positives in traditional environmental DNA (eDNA) metabarcoding analyses. Here, we alternatively employed environmental RNA (eRNA) metabarcoding to assess its potential for reducing false positive in ballast water monitoring. We collected eDNA and eRNA in parallel from ballast water before and after disinfection in three vessels. High-throughput sequencing of the 16S rRNA V4-V5 regions and cDNA counterparts was conducted to compare bacterial community composition. Our findings showed that over 80 % of the top 150 abundant amplicon sequence variants (ASVs) were detected by both eRNA and eDNA metabarcoding. Samples sequenced separately using DNA and RNA consistently clustered together, indicating similar community recovery efficacy. However, 42 % of ASVs were detected exclusively in DNA, resulting in significantly higher bacterial diversity compared to RNA, which suggests false positives in the DNA method. In treated samples with higher dead cell counts, the RNA method showed significantly lower bacterial diversity, indicating its effectiveness in detecting live bacteria. In summary, eRNA metabarcoding offers comparable recovery efficiency while maintaining a lower false-positive rate than eDNA metabarcoding.