Table 2.

Strategies used for optimization of next generation sequencing (NGS) microsatellite genotyping of Prochilodus costatus.

NGS Run	Strategy#	N° of primer pairs	N° of DNA samples	Cartridge¥	N° of filtered reads	∗∗N° of alleles
1	Multiplex	8, 4 and 2	5	1 M	13,691	∗
	Monoplex	8			829,631	∗
2	Multiplex	8	192	15 M	608,390	30
3	Pseudo-multiplex	8	192	1 M	486,130	49
4	Pseudo-multiplex	8	96	1 M	646,223	48
5	Pseudo-multiplex	8	288	1 M	1,051,375	52
6	Pseudo-multiplex	8	384	1 M	991,337	53

^∗Not applicate due to the low number of individuals. ^∗∗Total number of alleles obtained for eight loci and variable numbers of individuals. ^#Reactions used for amplification. Multiplex, in which all primer pairs were used to amplify a single DNA sample in a single reaction followed by the incorporation of the index and NGS sequencing; Pseudo-multiplex, in which each primer pair was individually used to amplify a DNA target, followed by pooling of the amplicons of one individual (eight loci per individual) and a second PCR for index incorporation and MiSeq sequencing; Monoplex, in which DNA target amplification and index incorporation were performed individually for each DNA target and followed by MiSeq sequencing. ^¥Type of cartridge used. 1 M is a MiSeq Reagent Nano Kit v2 (1 M reads) and 15 M is a MiSeq Reagent Kit v2 (15 M reads).