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 | 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 | 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 | 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 | |