Table 5.
Datasets used in the study and the corresponding experimental details
| Dataset name | Number of specimens | Fragment size, primer information | Extraction/PCR setup | PCR cleanup | ONT Library Preparation kit/Flow cell used |
|---|---|---|---|---|---|
| R10.3 Datasets | |||||
|
Mixed Diptera (see Srivathsan et al. [33]) - Sanger barcodes available |
511 (257 mixed Diptera, 254 Dolichopodidae) 17 negatives |
658 bp HCO2198, LCO1490 [80] |
Extraction Method: QuickExtract PCR Mix: Total volume: 20 μl 10× buffer: 2 μl dNTPs (2.5 mM): 1.5 μl Taq polymerase: 0.2 μl BSA (1 mg/ml): 2 μl Primer (5 μM): 2 μl each DNA:2 μl |
Ampure beads (Beckman Coulter) | SQK-LSK110/FLO-MIN111 |
|
Afrotropical Phoridae (see Srivathsan et al. [35]) - Illlumina mini-barcodes available |
4275 (Phoridae) 45 negatives |
658 bp HCO2198, LCO1490 [75] |
Extraction Method: QuickExtract PCR Mix: Total volume: 15.16 μl Mastermix (CWBio): 10 μl 25 mM MgCl2: 0.16 μl BSA (1 mg/ml): 2 μl Primer (10 μM): 1 μl each DNA: 1 μl |
Sera-Mag beads (GE Healthcare Life Sciences) in PEG | SQK-LSK109/FLO-MIN111 |
| Palaearctic Phoridae (658) |
9929 (Phoridae) 105 negatives |
658 bp jgHCO2198, LCO1490 [80, 81] |
Extraction Method: HotSHOT PCR Mix: Total volume: 16 μl Mastermix (CWBio): 7 μl BSA (1 mg/ml): 1 μl Primer (10 μM): 1 μl each DNA: 6 μl |
Ampure beads (Beckman Coulter) | SQK-LSK110/FLO-MIN111 |
| Palaearctic Phoridae (313) |
9932 (Phoridae) 106 negatives |
313 bp m1COlintF, jgHCO2198 [79, 81] |
Extraction Method: HotSHOT PCR Mix: Total volume: 14 μl Mastermix (CWBio): 7 μl BSA (1 mg/ml): 1 μl Primer (10 μM): 1 μl each DNA: 4 μl |
Ampure beads (Beckman Coulter) | SQK-LSK110/FLO-MIN111 |
| Flongle datasets | |||||
|
Mixed Diptera subsample (see Srivathsan et al. [33]) - Sanger barcodes available |
257 7 negatives |
See “Mixed Diptera” entry for R10.3 | See “Mixed Diptera” entry for R10.3 | Ampure beads (Beckman Coulter) | SQK-LSK109/Flongle |
| Chironomidae |
191 (Chironomidae) 1 negative |
313 bp m1COlintF, jgHCO2198 [74, 76] |
Extraction Method: HotSHOT PCR Mix: Total volume: 14 μl Mastermix (CWBio): 7 μl BSA (1 mg/ml): 1 μl Primer (10 μM): 1 μl each DNA: 4 μl |
Ampure beads (Beckman Coulter) | SQK-LSK109/Flongle |