Table 5. Amplicon size and number of reads (sequences) obtained for each of the three amplicons used for HTS.

Samples include three sets of constructed samples containing 3 replicates extracted using non-destructive (ND), complete homogenization (H) and dissection (D) based DNA extraction protocols, and six field-collected macroinvertebrate samples from three sites along Merri Creek (Melbourne, Australia) extracted using a non-destructive extraction protocol.

Sample (number of individuals)	Extraction method	LCOI/MLepR2 amplicon	B/COIBrev amplicon	mtCOIintF/HCOI amplicon		Overall species detection success (%)
		280 bp	293 bp	313 bp	Total
Samples of known composition:
Sample A (80)	ND	48,288	56,031	44,645	148,964	92
	H	117,667	116,313	61,850	295,830	84
	D	79,356	86,299	38,716	204,371	97
Sample B (100)	ND	67,448	75,433	19,463	162,344	89
	H	44,783	49,313	14,826	108,922	100
	D	111,207	124,103	68,281	303,591	100
Sample C (120)	ND	76,164	54,706	32,963	163,833	95
	H	39,418	65,326	24,561	129,305	95
	D	79,524	109,656	26,218	215,398	95
Sample C (120) technical replicate	ND	70,875	65,871	20,231	156,977	95
	H	86,622	134,934	24,233	245,789	100
	D	50,590	93,821	26,826	171,237	100
Field collected samples:
MRD 2016	ND	75,891	44,593	69,640	190,124	88
MRD 2014	ND	69,445	43,188	57,083	169,716	73
MOH 2016	ND	47,943	41,761	57,124	146,828	83
MOH 2014	ND	67,644	49,726	39,482	156,852	80
MCL 2016	ND	81,108	15,177	60,074	156,359	73
MCL 2014	ND	727	687	164	1,578	55 (low reads)