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Table 3 Comparison of barcodes obtained by ONTbarcoder with NGSpeciesID (green=highest number of correct and lowest number of errors for datasets). NGSpeciesID was applied once to the demultiplexed reads obtained with ONTbarcoder and once to those obtained with minibar. Barcode calling was done using all demultiplexed reads as well as 200X subset. ONTbarcoder was run at 200X read coverage (Consensus by Length) and 100X coverage (Consensus by Similarity). The accuracy of MinION barcodes is only compared to reference barcodes obtained with Illumina/Sanger sequencing. Errors are defined as the sum of substitution and indel errors

From: ONTbarcoder and MinION barcodes aid biodiversity discovery and identification by everyone, for everyone

  ONTBarcoder 200X ONTBarcoder dem1 + NGSpeciesID All reads ONTBarcoder dem1 + NGSpeciesID 200X Minibar + NGSpeciesID All reads Minibar + NGSpeciesID 200X
Mixed Diptera Subsample Flongle
Identical+compatible2(# of reference barcodes) 226 (231) 72 (241) 73 (241) 123 (241) 122 (241)
Identical* 177 70 71 121 120
Incorrect, (1/2/3/4/5+) errors 5 (3/1/1/0/0) 169 (83/60/13/6/7) 168 (88/55/13/7/5) 118 (29/21/8/4/56) 119 (31/20/8/4/56)
# reads demultiplexed/reads of length 658±50 33,270/32,978 19,511/15,935
Mixed Diptera R10.3
Identical+compatible2(# of reference barcodes) 474 (476) 173 (478) 353 (478) 418 (478) 453 (478)
Identical* 466 170 347 413 447
Incorrect, with (1/2/3/4/5+) errors 2 (0/0/1/0/1) 305 (126/97/48/17/17) 125 (91/24/5/1/4) 60 (45/9/3/0/3) 25 (20/2/0/0/3)
# reads demultiplexed/reads of length 658±50 1,544,768/1,532,408 1,540,751/1,378,910
Afrotropical Phoridae R10.3
Identical+compatible2 (# of reference barcodes) 3293 (3321) 2832 (3339) 3111 (3341) 3193 (3340) 3245 (3340)
Identical 3239 2832 3111 3193 3245
Incorrect, (1/2/3/4/5+) errors 28 (15/5/0/0/8) 507 (362/90/19/12/24) 230 (172/31/5/3/19) 147 (90/27/3/1/26) 95 (50/13/12/1/27)
# reads demultiplexed/reads of length 658±50 2,681,029/2,668,933 3,117,597/2,782,367
  1. 1dem = demultiplexing
  2. 2Identical barcodes match perfectly with references, and there are no ambiguities while compatible barcodes match with reference with at 100% identity but contain ambiguities. NGSpeciesID does not introduce N’s in barcodes
  3. *Compatible barcodes are found in NGSpeciesID datasets due to the presence of Ns in reference sequences