Dear EvolDir people, Many, many thanks to all that sent information and recommendations for my inquire about microsatellite primers commercially designed. I have summarized the information I received : (1) The Savannah River Ecology Laboratory in USA was mentioned several times and it really is the best price/product option. They use 454 sequencing following the enrichment step, producing a large number of primers : http://www.srel.edu/microsat/ Microsat_DNA_Development.html (2) Other companies were mentioned: Genetic ID Services, but they do not yet have 454 sequencing: http://www.genetic-id-services.com/ Microsynth (www.microsynth.ch) Genetic Marker Services, a microsatellite development service based in the UK (www.geneticmarkerservices.com). This one still has elevated prices (3) The general overview was to have 1/8 to 1/2 of a 454 plate (depending on the number of sequences-total primers needed) done through the commercial company of choice; doing multiple species is a good idea at not much extra cost. With the many (tousands) of reads (sequences) obtained, one can then perform 'at home' the identification and design of primers using free software (msatcommander the most recommended) and continue with the testing of polymorphism and amplification, as the cheapest way to go. See below: - You could have 1/4 of a plate of 454 done for each of the species, and then use msatcommander to identify and design primers. Macrogen has competitive prices on pyrosequencing, but shop around as many universities have a fee for service too, and may be cheaper. When you shop around, make sure they guarantee a certain number of sequences, or bases, as that will give you some indication as to how confident they are in their service. Contact them, and talk about your problem, I am sure you can get the sequencing done for a fraction of the 24K, and a 1/4 plate each species should yield you enough sequences to obtain at least 10 loci (I wouldn't be surprised if you managed to get more). - If you have no sequence data, you need to get that and there will be your biggest cost. We used 1/2 of a 454 plate using DNA from 2 individuals (1/4 plate each) (~$5000 a year ago but probably less now). You may be able to get away with 1/8 plate for each species if you only need 10 microsats. Once you have the sequence data, there are free programs that will search the sequence and identify repeats. Then, it is just a matter of testing them in several individuals to see if they are polymorphic. We had about a 60% success rate so if you designed 20 primer sets, you should get at least 10 good ones. I would guess that the costs would be closer to $7000 including the sequencing, primers, and testing. - We have used 454 sequencing and done the searches and primer design ourselves, which is quite easy with free software (msatcommander) and the searching process for candidate microsatellites took us about a week. The more time-consuming part is testing the primer pairs to ensure they amplify and then screening samples of individuals to assure polymorphism, allele size ranges, and lack of null alleles. Our initial 454-sequencing was conducted with several individuals combined in each DNA sample and and the three DNA samples were fragmented, coded and multiplexed. The sequencing cost was about $4100 USD and used 1/4 plate. We got approximately 250,000 reads with an average length between 400 and 500 bp and from this we were able to find several thousand candidate di-, tri-, and tetra-nucleotide microsatellites with forward and reverse primers. We ordered 40 primer pairs and about 75% of them amplify and so far about one- quarter of them microsatellites amplify in three putative species and are polymorphic in at least two of them - You could have 1/4 of a plate of 454 done for each of the species, and then use msatcommander to identify and design primers. Macrogen has competitive prices on pyrosequencing, but shop around as many universities have a fee for service too, and may be cheaper. When you shop around, make sure they guarantee a certain number of sequences, or bases, as that will give you some indication as to how confident they are in their service. Contact them, and talk about your problem, I am sure you can get the sequencing done for a fraction of the 24K, and a 1/4 plate each species should yield you enough sequences to obtain at least 10 loci (I wouldn't be surprised if you managed to get more). (4) Several papers were included that describe 454 sequencing used to develop microsatellite primers, which are of great help!! http://jhered.oxfordjournals.org/content/early/2010/06/18/ jhered.esq069.abstract http://www.biotechniques.com/BiotechniquesJournal/2009/March/ Identification-of-microsatellites-from-an-extinct-moa-species-using- high-throughput-454-sequence-data/biotechniques-118360.html http://www.biotechniques.com/BiotechniquesJournal/2009/March/ Microsatellite-discovery-by-deep-sequencing-of-enriched-genomic- libraries/biotechniques-118349.html http://jhered.oxfordjournals.org/content/early/2010/07/11/ jhered.esq080.full http://sequence.otago.ac.nz/download/454_FAQ.pdf  Original post in EvolDir: Hello, I need to have primers developed from a commercial/educational company. In the best of worlds I would like to have 10 polymorphic microsatellite primers for each of two mammal species. I have one quote, from Ecogenics, that includes 454 sequencing enriched for two species (7,700 us dlls) and the 10 polymorphic loci per species (825 per locus, which totals to 16,500 dlls, for a grand total of 24,250). This sounds no doubt like a great service and product deliver, but very far from the budget I have. Has anyone have primers developed and/or could suggest companies to contact? I will really appreciate any help. Thanks a million, Ella evazquez@ecologia.unam.mx