Table 1.

Datasets generated in this study and the results of barcoding using ONTbarcoder at 200X coverage (Consensus by Length) and 100X coverage (Consensus by Similarity)

Dataset name	Flow cell details Run time/Guppy version	Raw reads/reads passing length threshold/reads of suitable length/ demultiplexed	Demultiplexing rate/# QC_compliant barcodes/# Filtered barcodes with 1 N/# Filtered barcodes with > 1 N /# Unreliable barcodes
MinION R10.3 datasets
Mixed Diptera (658 bp, N=511)	R10.3: reused flow cell: 71 pores according to QC, but 500+ active during run Runtime: 27.5 h Guppy: 4.2.3+f90bd04	3,864,000/3,425,357/3,560,389/1,544,758	43.39%/495/2/5/8 Total success rate= 502/511 (98.2%)
Afrotropical Phoridae (658 bp, N=4275)	R10.3: new flow cell: QC: 1101 pores Runtime: 49.5 h Guppy: 4.0.11+f1071ce	6,838,903/5,465,164/5,474,306/2,681,029	48.97%/3722/121/59/247 Total success rate= 3905/4275 (91.3%)
Palaearctic Phoridae (658 bp, N=9932)	R10.3: new flow cell: QC: 1239 pores Runtime: 47.5 h Guppy: 4.2.3+f90bd04	16,595,984/15,658,174/16,100,505/5,012,489	31.13%/8026/108/231/780 Total success rate= 8365/9932 (84.2%)
Palaearctic Phoridae (313 bp, N=9929)	R10.3: new flow cell: QC: 1297 pores Runtime: 37 h Guppy: 4.2.3+f90bd04	13,690,869/13,221,764/10,366,455/12,983,260/2,015,135	15.52%/8705/118/112/899 Total success rate= 8935/9929 (90%)
Flongle datasets
Mixed Diptera Subsample (658 bp, N=257)	Flongle: new QC: 81 pores Runtime: 24 h Guppy: v 4.0.11+f1071ce	294,896/222,189/190,952/33,270	17.42%/185/35/20/9 Total success rate= 240/257 (93.4%)
Chironomidae (313 bp, N=191)	Flongle: new QC: 74 pores Runtime: 15 h Guppy: 4.2.3+f90bd04	560,062/525,087/504,621/108,574	21.52%/178/1/2/6 Total success rate= 181/191 (94.8%)