I would consider a RAD-seq type approach using a MiSeq 2x300 run on a limited number of samples. The 2x300 run would give you >500 bp of contiguous sequence for analysis, RAD would reduce the complexity allowing you to oversample a limited number of loci (50,000 for example) for high quality analysis and yet provide enough sequence space to identify microsats that are polymorphic between samples.

Hello Kirsten

Are you absolutely certain that microsatellites are the best possible marker for your study? In other words, what are the goals of your study, and what is the DNA quality and quantity of the material you will be starting from? Will this study be the start of some long-term monitoring (>5 years)? Monitoring with microsats will always be faced to problems with allele-calling across sequencing machines and platforms. 

I'm asking these questions because microsatellites are not exactly cheap to develop and you get only a limited amount of useful markers (typically just 10-20 markers within an optimized protocol for 10 000 euro). So how many markers do you need for your approach? 

I've recently compared different approaches for cost-efficiency: microsat development based on 454 reads (a few years ago) and RAD reads and genotyping in the own lab, SNP development based on RAD-reads (Floragenex) and outsourcing the genotyping (KASP by LGC Genomics), and genotyping by sequencing (GBS, outsourced to Cornell University Institute for Genomic Diversity). 

I compared 15 microsats to 60 SNPs (about the same resolution for genotyping as 15 microsats), to 150 SNPs and to GBS (typically 2000-5000 SNPs). My conclusion was that below 400 samples, GBS is always the cheapest, followed by 60 SNPs, and then microsats. Microsats only became cheaper than GBS at around 1000 samples, but the difference in resolution is huge! No matter how many samples you analyze, 60 SNPs are always cheaper to genotype than 15 microsats.

The downside of GBS and to a lesser extent of SNPs is that these methods require large amounts of DNA of rather high quality. So if you need to do non-invasive sampling (e.g., skin or mouth swabs in amphibians) GBS is not an option (you would need to cut a toe or similar), and microsats are the most feasible.

Say DNA is not an issue and you have more than 1000 samples, should you use 60 SNPs or 15 microsats? That depends on the research question. Does your focal species (or a very closely related species) have a published draft genome? Are you interested in identifying genomic adaptation? If you are mostly interested in the phylogeography of a species in a region, microsats (with stepwise mutation models) may be the better option (but 15 markers is very meager then). But if you are not interested in phylogeographic patterns, stepwise mutation models are a nuisance rather than an advantage (see my answer to this post: https://www.researchgate.net/post/How_many_microsatellite_loci_and_what_sample_size_should_be_used_in_population_differentiation?_tpcectx=profile_answers)

For papers using the different methods of marker development, see for example

SNPs: https://www.researchgate.net/publication/258277159_Isolation_characterization_and_genotyping_of_single_nucleotide_polymorphisms_in_the_non-model_tree_species_Frangula_alnus_%28Rhamnaceae%29?ev=prf_pub

SNPs & microsats https://www.researchgate.net/publication/233900188_SNP_discovery_using_Paired-End_RAD-tag_sequencing_on_pooled_genomic_DNA_of_Sisymbrium_austriacum_%28Brassicaceae%29?ev=prf_pub

https://www.researchgate.net/publication/235673695_Microsatellite_marker_development_and_putative_SNP_detection_for_a_northward_expanding_damselfly_species_using_next_generation_sequencing?ev=prf_pub

GBS: https://www.researchgate.net/publication/262645659_Landscape_genomics_and_a_common_garden_trial_reveal_adaptive_differentiation_to_temperature_across_Europe_in_the_tree_species_Alnus_glutinosa?ev=prf_pub

Article Isolation, characterization and genotyping of single nucleot...

Article SNP discovery using Paired-End RAD-tag sequencing on pooled ...

Article Microsatellite marker development and putative SNP detection...

Article Landscape genomics and a common garden trial reveal adaptive...

Ecogenics

Hi Kirsten,

I completely agree with Joachim (btw, thank you for this exhaustive comparison).

However, if you still decide to go for microsatellites, I recommend Genoscreen (France). They are the best people do develop microsat using NGS since they develop the technique: https://www.researchgate.net/publication/51221963_High-throughput_microsatellite_isolation_through_454_GS-FLX_Titanium_pyrosequencing_of_enriched_DNA_libraries?ev=prf_pub

From the calculations I made last time I developed microsat de novo, you can obtain quite easily 20-25 markers for ca 5000€. In this case, you would need to perform some steps by yourself, but it is very simple.

I personally used the services of Genoscreen for developing microsat on plants and fungi. If you need more advices about the procedure, do not hesitate to contact me.

Article High-throughput microsatellite isolation through 454 GS-FLX ...

Hi All

Thank you all for your input. Yes, Joachim, thanks especially for your comparison. I think that we will be dealing with under 500 samples and we really need to check that microsatellites will be the most cost effective and informative direction to take.

Our samples will be small, but fresh skin preserved in ethanol, so the DNA should be of high quality after extraction.

Kirsten

Hi Kirsten, we have used Genoscreen and Ecogenics for our microsats so far and were very satisfied with them - they have a stepwise pricing system, allowing you to get products at every stage of the process, so for example, you could get the full library they can obtain, and then do the polymorphy check for yourself.