A single degenerated primer significantly improves COX1 barcoding performance in soil nematode community profiling

• A new COX1 primer for soil nematode metabarcoding was designed, and this primer outperforms other commonly used COX1 primer pairs in species recovery and quantity of PCR products. • The lack of reference database is the main reason that led to the low species recovery in COX1 metabarcoding. • We expanded current NCBI database by adding 51 newly generated COX1 reference sequences. Microscopic nematodes play important roles in soil ecosystems and often serve as bioindicators of soil health. The identification of soil nematodes is often difficult due to their limited diagnostic characters and high phenotypic plasticity. DNA barcoding and metabarcoding techniques are promising but lack universal primers, especially for mitochondrial COX1 gene. In this study a degenerated COX1 forward primer COIFGED was developed. The primer pair (COIFGED/JB5GED) outperforms other four commonly used COX1 primer pairs in species recovery and quantity of polymerase chain reaction (PCR) products. In metabarcoding analysis, the reads obtained from the new primer pair had the highest sequencing saturation threshold and amplicon sequence variant (ASV) diversity in comparison to other COX1 as well as 18S rRNA primers. The annotation of ASVs suggested the new primer pair initially recovered 9 and 6 out of 25 genera from mock communities, respectively, outperformed other COX1 primers, but underperformed the widely used 18S NF1/18Sr2b primers (16 out of 25 genera). By supplementing the COX1 database with our reference sequences, we recovered an additional 6 mock community species bringing the tally closer to that obtained with 18S primers. In summary, our newly designed COX1 primers significantly improved species recovery and thus can be supplementary or alternative to the conventional 18S metabarcoding.