Deoxyribonucleic acid extraction for invertebrate metabarcoding from stream sediment and implication to biotic indices

BACKGROUND AND OBJECTIVES: Environmental deoxyribonucleic acid metabarcoding has become a powerful tool for assessing invertebrate biodiversity in stream sediments, providing a non-invasive approach for ecological monitoring. However, the accuracy of this method heavily depends on the nucleic acid extraction protocol used, which can influence taxonomic recovery and the resulting biotic indices. Previous studies have focused on nucleic acid yield and taxonomic composition, but limited research has evaluated the impact of extraction methods on ecological assessments. This study aims to compare four nucleic acid extraction protocols to determine their effectiveness in maximizing metazoan recovery and improving the reliability of biotic indices derived from metabarcoding data.METHODS: Four nucleic acid extraction protocols were tested for invertebrate nucleic acid metabarcoding from stream sediment: a manual extraction and three Qiagen commercial kits (Powersoil, Blood and Tissue, and Plant Mini kit). Extracted nucleic acid was amplified using cytochrome c oxidase I and 18 Svedberg ribosomal ribonucleic acid hypervariable 4 and 9 markers and sequenced using Illumina sequencing. The relative read abundance was analyzed to assess metazoan recovery and biotic index scores were calculated based metazoan community of respective extraction protocol.FINDINGS: Among the tested methods, PowerSoil kit consistently yielded the lowest metazoan relative read abundance (36.07 to 86.31 percent) from stream sediment and exhibited higher non-target amplification, including bacterial nucleic acid, due to its bead-beating step optimized for prokaryotic nucleic acid extraction. In contrast, other protocols showed superior metazoan recovery (63.83 to 98.58 percent) and minimized bacterial co-amplification, indicating high specificity for eukaryotic nucleic acid. Plant Mini kit performed unexpectedly well, aligning closely with the manual extraction in biotic indices, likely due to its effective inhibitor removal of amplification. Conversely, blood and tissue kit had the highest dissimilarity in biotic indices, potentially attributed to the inhibition, resulting in lower detection of sensitive taxa. Biotic indices derived from cytochrome c oxidase I and 18 Svedberg ribosomal ribonucleic acid hypervariable 9 markers were comparable, with cytochrome c oxidase I showing slightly higher scores due to its greater specificity in targeting key taxa used for index calculation. The 18 Svedberg ribosomal ribonucleic acid hypervariable 4 marker, however, yielded lower biotic index scores and was less effective in metazoan recovery, particularly for invertebrates sensitive to ecological stressors.CONCLUSION: Overall, the choice of extraction method substantially influenced the detection of target metazoan taxa and the reliability of biotic indices, with the manual extraction and Plant Mini kit emerging as the most effective protocols. These findings underscore the importance of protocol selection in metabarcoding of stream sediments, suggesting that optimized methods tailored for metazoan nucleic acid recovery are crucial for accurate ecological assessments.