Metabarcoding combines DNA barcoding with high-throughput sequencing, often using one genetic marker to understand complex and taxonomically diverse samples. However, species-level identification depends heavily on the choice of marker and the selected primer pair, often with a trade-off between successful species amplification and taxonomic resolution. We present a versatile metabarcoding protocol for biomonitoring that involves the use of two barcode markers (COI and 18S) and four primer pairs in a single high-throughput sequencing run, via sample multiplexing. We validate the protocol using a series of 24 mock zooplanktonic communities incorporating various levels of genetic variation. With the use of a single marker and single primer pair, the highest species recovery was 77%. With all three COI fragments, we detected 62-83% of species across the mock communities, while the use of the 18S fragment alone resulted in the detection of 73-75% of species. The species detection level was significantly improved to 89-93% when both markers were used. Furthermore, multiplexing did not have a negative impact on the proportion of reads assigned to each species and the total number of species detected was similar to when markers were sequenced alone. Overall, our metabarcoding approach utilizing two barcode markers and multiple primer pairs per barcode improved species detection rates over a single marker/primer pair by 14% to 35%, making it an attractive and relatively cost-effective method for biomonitoring natural zooplankton communities. We strongly recommend combining evolutionary independent markers and, when necessary, multiple primer pairs per marker to increase species detection (i.e. reduce false negatives) in metabarcoding studies.
1a_R1.fastq
NGS library 1a R1 raw reads
MI.M03555_0088.001.N701---S502.1a_R1.fastq.gz
1a_R2.fastq
NGS library 1a R2 raw reads
MI.M03555_0088.001.N701---S502.1a_R2.fastq.gz
2a_R1.fastq
NGS library 2a R1 raw reads
MI.M03555_0088.001.N701---S503.2a_R1.fastq.gz
2a_R2.fastq
NGS library 2a R2 raw reads
MI.M03555_0088.001.N701---S503.2a_R2.fastq.gz
3a1_R1.fastq
NGS library 3a1 R1 raw reads (Limnoperna fortunei; n=1)
MI.M03555_0088.001.N701---S504.1xL-fort_R1.fastq.gz
3a1_R2.fastq
NGS library 3a1 R2 raw reads (Limnoperna fortunei; n=1)
MI.M03555_0088.001.N701---S504.1xL-fort_R2.fastq.gz
3c1_R1.fastq
NGS library 3c1 R1 raw reads (Tortanus discaudatus; n=1)
MI.M03555_0088.001.N701---S517.1xT-discau_R1.fastq.gz
3c1_R2.fastq
NGS library 3c1 R2 raw reads (Tortanus discaudatus; n=1)
MI.M03555_0088.001.N701---S517.1xT-discau_R2.fastq.gz
1b_R1.fastq
NGS library 1b R1 raw reads
MI.M03555_0088.001.N702---S502.1b_R1.fastq.gz
1b_R2.fastq
NGS library 1b R2 raw reads
MI.M03555_0088.001.N702---S502.1b_R2.fastq.gz
2b_R1.fastq
NGS library 2b R1 raw reads
MI.M03555_0088.001.N702---S503.2b_R1.fastq.gz
2b_R2.fastq
NGS library 2b R2 raw reads
MI.M03555_0088.001.N702---S503.2b_R2.fastq.gz
3a2_R1.fastq
NGS library 3a2 R1 raw reads (Limnoperna fortunei; n=10)
MI.M03555_0088.001.N702---S504.10xL-fort_R1.fastq.gz
3a2_R2.fastq
NGS library 3a2 R2 raw reads (Limnoperna fortunei; n=10)
MI.M03555_0088.001.N702---S504.10xL-fort_R2.fastq.gz
3c2_R1.fastq
NGS library 3c2 R1 raw reads (Tortanus discaudatus; n=8)
MI.M03555_0088.001.N702---S517.8xT-discau_R1.fastq.gz
3c2_R2.fastq
NGS library 3c2 R2 raw reads (Tortanus discaudatus; n=8)
MI.M03555_0088.001.N702---S517.8xT-discau_R2.fastq.gz
1c_R1.fastq
NGS library 1c R1 raw reads
MI.M03555_0088.001.N703---S502.1c_R1.fastq.gz
1c_R2.fastq
NGS library 1c R2 raw reads
MI.M03555_0088.001.N703---S502.1c_R2.fastq.gz
2c_R1.fastq
NGS library 2c R1 raw reads
MI.M03555_0088.001.N703---S503.2c_R1.fastq.gz
2c_R2.fastq
NGS library 2c R2 raw reads
MI.M03555_0088.001.N703---S503.2c_R2.fastq.gz
3a3_R1.fastq
NGS library 3a3 R1 raw reads (Limnoperna fortunei; n=30)
MI.M03555_0088.001.N703---S504.30xL-fort_R1.fastq.gz
3a3_R2.fastq
NGS library 3a3 R2 raw reads (Limnoperna fortunei; n=30)
MI.M03555_0088.001.N703---S504.30xL-fort_R2.fastq.gz
3c3_R1.fastq
NGS library 3c3 R1 raw reads (Tortanus discaudatus; n=15)
MI.M03555_0088.001.N703---S517.15xT-discau_R1.fastq.gz
3c3_R2.fastq
NGS library 3c3 R2 raw reads (Tortanus discaudatus; n=15)
MI.M03555_0088.001.N703---S517.15xT-discau_R2.fastq.gz
1d_R1.fastq
NGS library 1d R1 raw reads
MI.M03555_0088.001.N704---S502.1d_R1.fastq.gz
1d_R2.fastq
NGS library 1d R2 raw reads
MI.M03555_0088.001.N704---S502.1d_R2.fastq.gz
2d_R1.fastq
NGS library 2d R1 raw reads
MI.M03555_0088.001.N704---S503.2d_R1.fastq.gz
2d_R2.fastq
NGS library 2d R2 raw reads
MI.M03555_0088.001.N704---S503.2d_R2.fastq.gz
3b1_R1.fastq
NGS library 3b1 R1 raw reads (Balanus crenatus; n=1)
MI.M03555_0088.001.N704---S504.1xB-cre_R1.fastq.gz
3b1_R2.fastq
NGS library 3b1 R2 raw reads (Balanus crenatus; n=1)
MI.M03555_0088.001.N704---S504.1xB-cre_R2.fastq.gz
3d1_R1.fastq
NGS library 3d1 R1 raw reads (Leptodora kindtii; n=1)
MI.M03555_0088.001.N704---S517.1xL-kind_R1.fastq.gz
3d1_R2.fastq
NGS library 3d1 R2 raw reads (Leptodora kindtii; n=1)
MI.M03555_0088.001.N704---S517.1xL-kind_R2.fastq.gz
1e_R1.fastq
NGS library 1e R1 raw reads
MI.M03555_0088.001.N705---S502.1E_R1.fastq.gz
1e_R2.fastq
NGS library 1e R2 raw reads
MI.M03555_0088.001.N705---S502.1E_R2.fastq.gz
2e_R1.fastq
NGS library 2e R1 raw reads
MI.M03555_0088.001.N705---S503.2E_R1.fastq.gz
2e_R2.fastq
NGS library 2e R2 raw reads
MI.M03555_0088.001.N705---S503.2E_R2.fastq.gz
3b2_R1.fastq
NGS library 3b2 R1 raw reads (Balanus crenatus; n=10)
MI.M03555_0088.001.N705---S504.10xB-cre_R1.fastq.gz
3b2_R2.fastq
NGS library 3b2 R2 raw reads (Balanus crenatus; n=10)
MI.M03555_0088.001.N705---S504.10xB-cre_R2.fastq.gz
3d2_R1.fastq
NGS library 3d2 R1 raw reads (Leptodora kindtii; n=10)
MI.M03555_0088.001.N705---S517.10xL-kind_R1.fastq.gz
3d2_R2.fastq
NGS library 3d2 R2 raw reads (Leptodora kindtii; n=10)
MI.M03555_0088.001.N705---S517.10xL-kind_R2.fastq.gz
1g_R1.fastq
NGS library 1g R1 raw reads
MI.M03555_0088.001.N706---S502.1G_R1.fastq.gz
1g_R2.fastq
NGS library 1g R2 raw reads
MI.M03555_0088.001.N706---S502.1G_R2.fastq.gz
2g_R1.fastq
NGS library 2g R1 raw reads
MI.M03555_0088.001.N706---S503.2G_R1.fastq.gz
2g_R2.fastq
NGS library 2g R2 raw reads
MI.M03555_0088.001.N706---S503.2G_R2.fastq.gz
3b3_R1.fastq
NGS library 3b3 R1 raw reads (Balanus crenatus; n=17)
MI.M03555_0088.001.N706---S504.17xB-cre_R1.fastq.gz
3b3_R2.fastq
NGS library 3b3 R2 raw reads (Balanus crenatus; n=17)
MI.M03555_0088.001.N706---S504.17xB-cre_R2.fastq.gz
3d3_R1.fastq
NGS library 3d3 R1 raw reads (Leptodora kindtii; n=28)
MI.M03555_0088.001.N706---S517.28xL-kind_R1.fastq.gz
3d3_R2.fastq
NGS library 3d3 R2 raw reads (Leptodora kindtii; n=28)
MI.M03555_0088.001.N706---S517.28xL-kind_R2.fastq.gz
Sanger Sequences from this Study
Sanger sequences generated in this study, included in the local database as reference sequences
SangerSequencesThisStudy.fasta