Table 1.

Case studies of microsatellite development using the described pipeline

Species	P	No. reads (2×)		No. loci with primers [total no. loci]		ST [SG] (%)
		Raw	Filtered	Raw reads	Filtered reads
Amietia hymenopus (Phofung river frog)	0.5	6,465,564	3,756,407	25,427 [149,271]* 1,345† 216‡	11,350 [60,378]* 1097† 144‡	56 [64]
Raja undulata (Undulate ray)	0.5	11,019,590	10,174,420	267,431 [130,894]* 3119† 428‡	107,470 [31,876]* 342† 148‡	73 [80]
Modiolus modiolus (Northern horsemussel)	0.125	4,647,211	4,455,417	64,489 [44,408]* 1650† 225‡	39,232 [16,814]* 707† 144‡	53 [74]

All sequencing was paired end, carried out on the Illumina MiSeq, with sequence lengths of 2 × 250 bp. Trimmomatic settings (SLIDING WINDOW: WINDOW SIZE = 4 bp, QUALITY = 20; LEADING = 3; TRAILING = 3; MINLEN = 50) and primer design conditions (recommended settings for Qiagen Type-it^® Microsatellite PCR kit) were constant across all tests. Minimum number of microsatellite repeats to be searched for was eight for all repeat types (2-6mer)

P, proportion of Illumina flow cell lane used; * without pal_filter or assembly; ^† with pal_filter (all filtering options selected), without assembly; ^‡ with pal_filter (all filtering options selected) and assembly; S_T, total amplification success rate − percentage of loci tested that resulted in amplifiable loci that could be easily scored when fluorescently labeled and analysed using an automated capillary sequencer; S_G, amplification success rate using agarose gel electrophoresis − percentage of loci tested that resulted in clear bands when visualising PCR products of unlabeled primers on an agarose gel. Primers used in this test were developed from Trimmomatic-fitered reads, with all of the pal_filter and assembly options selected