From karenf_at_camden.usyd.edu.au Sun Oct 9 19:33:34 2005 Date: Mon, 10 Oct 2005 10:35:20 +1000 From: Karen Fullard <karenf_at_camden.usyd.edu.au> Subject: Experiences with Genotypers To: Multiple Recipients of <angenmap_at_animalgenome.org> Dear GeneMappers, We are a medium throughput research laboratory with a number of diverse users from postgraduate students to dedicated personnel on mapping and QTL projects. We are looking to increase our in-house genotyping capacity (microsats, SNPs, possibly SSCP). Before spending a large amount of money, we thought it would be a good idea to canvass people's experience of using the high throughput ABI, MegaBace and Beckman platforms. Any comparisons/feedback on reliability, ease of use (including software), cost, throughput or anything else of relevance would be greatly appreciated. Thanks in advance, Karen Fullard

From genhua_at_tll.org.sg Sun Oct 9 21:37:30 2005 From: "Gen Hua" <genhua_at_tll.org.sg> References: <bsXuOD.A.nHF.fbbSDB_at_nagrp.ansci.iastate.edu> Subject: Re: Experiences with Genotypers Date: Mon, 10 Oct 2005 10:37:22 +0800 To: Multiple Recipients of <angenmap_at_animalgenome.org> Dear Karen Fullard, We are using the ABI3730xl for large scale genotyping of fish samples. In the last 12 months we gonotyped 50,000 fish individuals with 9-19 microsatellite markers, we also genotyped a mapping panel including 96 Asian seabass individuals with 250 microsatellites. Although the ABI3730xl is quite expensive, it is reliable and high throughput. The 51cm capillary can be used for microsatellite genotyping, AFLP analysis and DNA sequencing. The software GeneMapper V3.5 is also very good. If you need more information, you may contact me. Regards Gen Hua YUE (Ph.D) Molecular Population Genetics Group Manager Strategic Research Program Temasek Life Sciences Laboratory 117604 Singapore Tel: 65-68727405 Fax: 65-68727007 E-mail: genhua_at_tll.org.sg

From taylorjerr_at_missouri.edu Mon Oct 10 09:32:58 2005 Subject: RE: Experiences with Genotypers Date: Mon, 10 Oct 2005 09:32:53 -0500 From: "Taylor, Jerry F." <taylorjerr_at_missouri.edu> Return-Path: taylorjerr_at_missouri.edu To: Multiple Recipients of <angenmap_at_animalgenome.org> Hi Karen: The answers to your questions depend quite a lot on context. My lab is currently running about a million bovine microsatellite genotypes a year to perform genome scans, to asign parentage and to detect hybridization between cattle and bison. We have developed multiplexes for about 400 loci for genome scans and average 6-7 loci in each mutliplex PCR. The parentage and purity sets are 17 and 11 loci respectively and are each run in two multiplex PCRs. We detect the markers on an ABI 3730XL or ABI 3100 and score genotypes using GeneMapper. I am not a major fan of the ABI software, but it gets the job done after you learn how to use it. The instruments are now reliable and the 3730 has enormous capacity. Ignoring the labor and primer cost, we can produce microsatellite genotypes for 20 cents each. However, there's a fair amount of work invested in developing the multiplexes and if you want to run a specific set of 6-7 markers in one multiplex, you may have to redesign primers. However, considering that the information content of a microsatellite is 3-4x that of a SNP, the cost is very competitive. The big problem is the time and labor cost. For a genome scan we need to run 60-70 multiplex reactions and this takes weeks. We have just acquired an Illumina BeadStation for our whole genome linkage and linkage disequilibrium work. I have been collaborating on SNP validation with a colleage in Canada who has one and it takes 2 days to run an assay which produces 1536 SNP genotypes on a single sample. There are other platforms that will detect more SNPs in a single assay, but you need to figure out what is optimal for your application. We think that 1536 is fine for primary linkage and 3072 should be OK for LD studies in cattle where there is long range LD. If you score 1536 SNPs in 960 samples, the cost works out to be 5.6 cents per SNP. For projects requiring scoring smaller numbers of SNPs, we looked at the Beckman SNPstream and decided against the instrument due to cost and the fact that you currently need to score all the same types of SNPs (i.e. C/T) within a multiplex. This reduces flexibility and increases cost (Beckman says about 10 cents a SNP). A colleage here at Mizzou just bought a Luminex 100 for this purpose. The instrument is inexpensive (relative to the Beckman) and allows the theoretical multiplexing of up to 100 SNPs in an assay. I don't know anyone that has achieved that, but I do know someone that's running 70 SNPs (in two reactions that are pooled). Hope this is useful. Jerry *********************************************************** Jeremy Taylor Professor and Wurdack Chair in Animal Genomics S135 ASRC University of Missouri 920 East Campus Drive Columbia, MO 65211-5300 Voice: (573) 884-4946 Fax: (573) 882-6827 E-mail: taylorjerr_at_missouri.edu http://animalgenomics.missouri.edu

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