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