Open Access Method2005ZipperlenetVolume al. 6, Issue 2, Article R19 A universal method for automated gene mapping comment Peder Zipperlen¤*, Knud Nairz¤†, Ivo Rimann†, Konrad Basler*, Ernst Hafen†, Michael Hengartner* and Alex Hajnal† Addresses: *Institute of Molecular Biology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. †Institute of Zoology, University of Zürich, Winterthurerstrasse 190, CH-8057 Zürich, Switzerland. ¤ These authors contributed equally to this work. reviews Correspondence: Peder Zipperlen. E-mail: [email protected]. Knud Nairz. E-mail: [email protected] Published: 17 January 2005 Received: 9 September 2004 Revised: 15 November 2004 Genome Biology 2005, 6:R19 Accepted: 9 December 2004 The electronic version of this article is the complete one and can be found online at http://genomebiology.com/2005/6/2/R19 reports © 2005 Zipperlen et al.; licensee BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. Mapping<p>Amaps forhigh-throughput DrosophilaInDel sequence and method C.polymorphisms. elegans.</p> for genotyping by mapping InDels. This method has been used to create fragment-length polymorphism deposited research Abstract Small insertions or deletions (InDels) constitute a ubiquituous class of sequence polymorphisms found in eukaryotic genomes. Here, we present an automated high-throughput genotyping method that relies on the detection of fragment-length polymorphisms (FLPs) caused by InDels. The protocol utilizes standard sequencers and genotyping software. We have established genome-wide FLP maps for both Caenorhabditis elegans and Drosophila melanogaster that facilitate genetic mapping with a minimum of manual input and at comparatively low cost. research refereed Background where allele-specific, dual-labeled fluorescent TaqMan For humans and model organisms, such as worms and flies, probes guarantee specificity [7]. However, the need for two the availability of high-density sequence polymorphism maps dual-labeled fluorescent probes, expensive specialized chem- greatly facilitates the rapid mapping and cloning of genes [1- istry and specialized machinery increase the costs per assay of 3]. Key advantages of most molecular polymorphisms are the this approach significantly. Similarly, denaturing high-per- fact that they are codominant and in general phenotypically formance liquid chromatography (DHPLC) also analyses the interactions neutral. The vast majority of sequence polymorphisms are primary amplification product [8]. This approach is based on single-nucleotide polymorphisms (SNPs). melting differences of homo- versus heteroduplex DNA frag- ments under increasingly denaturing conditions and requires The most direct approach for SNP detection is sequencing of no specific labeling of the PCR fragments. However, condi- a PCR product spanning the polymorphism, but this is too tions have to be optimized for every assay, throughput is lim- costly and labor intense for high-throughput genotyping. For ited and specialized equipment is required. DHPLC has been this reason, several different strategies and methods have used in small-scale genotyping projects in Drosophila mela- been developed in order to detect SNPs more efficiently. In nogaster [9]. information general, assays can be grouped into strategies, where the nature of the SNP is determined by directly analyzing the pri- Of the methods that detect the SNP in a secondary assay, mary PCR product and those that require a secondary assay restriction fragment length polymorphism (RFLP) analysis performed on the primary amplification product [4-6]. An are very popular [10]. For this purpose, only those SNPs that important strategy of the first group is the 5' nuclease assay, alter a restriction site are analyzed. A great advantage of Genome Biology 2005, 6:R19 R19.2 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. http://genomebiology.com/2005/6/2/R19 (a) ZH1-01: 3bp InDel; no A addition (b) ZH2-01: 12bp InDel; with A addition (c) ZH3-05a: 2bp InDel; with A addition Fragment length (bp) Fragment length (bp) Fragment length (bp) 117118 119 120 121 122 123 134 136 138 140 142 144 146 148 150 174175 176 177 178 179 180 1600 2000 6000 1600 1200 4000 1200 800 800 2000 400 400 Bristol Bristol Bristol 117118 119 120 121 122 123 134 136 138 140 142 144 146 148 150 [bp] 174175 176 177 178 179 180 300 12000 4000 10000 3000 8000 200 6000 2000 4000 100 1000 2000 Flourescence Hawaii Hawaii Hawaii 117118 119 120 121 122 123 134 136 138 140 142 144 146 148 150 [bp] 174175 176 177 178 179 180 600 5000 4000 4000 400 3000 3000 2000 2000 200 1000 1000 Bristol Hawaii Bristol Hawaii Bristol Hawaii (d) ZH3-23: 1bp InDel; with A addition (e) 3R160: 1bp InDel; poly-T stretch (f) ZHX-22: 6bp InDel; poly-C stretch Fragment length (bp) Fragment length (bp) Fragment length (bp) 120 125 130 171172 173 174 175 176 177 201 203 205 207 209 211 213 6000 3000 12000 5000 10000 4000 2000 8000 3000 6000 1000 4000 2000 1000 1 2 2000 1 2 3 Bristol EP Bristol 120 125 130 171172 173 174 175 176 177 201 203 205 207 209 211 213 5000 14000 10000 12000 4000 8000 10000 3000 8000 6000 2000 6000 4000 4000 2000 1000 2000 Flourescence 1 2 1 2 3 Hawaii FRT Hawaii 120 125 130 171172 173 174 175 176 177 201 203 205 207 209 211 213 10000 20000 5000 16000 8000 4000 12000 6000 3000 8000 4000 2000 4000 2000 1000 1 2 3 1 2 3 4 Bristol Hawaii EP FRT Hawaii Bristol FLPFigure detection 1 of InDels of various sizes in homozygotes and heterozygotes FLP detection of InDels of various sizes in homozygotes and heterozygotes. In each panel the top two graphs show the homozygotes and the bottom graph the heterozygote. Gray shaded areas mark the defined expected allele lengths and red lines indicate the borders of a predefined window of expected allele lengths. (a-c) Detection of InDels in C. elegans that show increasing levels of adenosine (A) addition. (a) 3-bp InDel ZH1-01 with no A addition; (b) 12-bp InDel ZH2-01 with A addition; (c) 2-bp InDel ZH3-05a with A addition. (d) 1-bp InDel ZH3-23 in C. elegans with A addition. An unambiguous allele- call can be made, irrespectively of the level of A addition: both homozygous samples consist of two peaks at different positions, whereas the heterozygous animal exhibits three peaks. (e) The 1-bp InDel 3R160 in Drosophila runs over a 12-13 nucleotide poly(T) stretch and exhibits stutter bands. Even in this case, a clear allele-call can be made (three peaks in homozygous and four peaks in heterozygous animals). (f) The 6-bp InDel ZHX-22 in C. elegans occurs in a poly(C) stretch and the FLP graph displays stutter bands. As expected, the longer fragment exhibits a higher degree of stuttering. Genome Biology 2005, 6:R19 http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. R19.3 RFLP analysis is that no specialized equipment is needed and formed automatically using the Applied Biosystems GeneMa- it can be carried out in every laboratory. RFLP maps recently pper software commonly used for genotyping STRs established for Caenorhabditis elegans and Drosophila are (Materials and methods). comment used regularly in genotyping projects [2,3,11]. However, RFLP analysis requires significant manual input. Moreover, To demonstrate the feasibility of this strategy, we have vali- the use of different restriction enzymes with different reac- dated 112 evenly spaced FLP assays at 3 centimorgan (cM) tion requirements adds another level of complexity that resolution in C. elegans (one every 0.9 megabase-pair (Mbp)) makes this method difficult to automate. Primer-extension- and 54 FLP assays at 4 cM resolution for the Drosophila auto- based technologies have also gained some prominence [12]. somes. This set of FLP assays allows us to rapidly map muta- Here, a primer that anneals right next to the polymorphism is tions to small chromosomal subregions with a minimum of extended by one polymorphism-specific terminator nucle- manual input. Furthermore, we provide a list of predicted reviews otide. Extension products are analyzed by size or, alterna- InDels for which additional assays can be readily designed in tively, by differences in the behavior of incorporated versus the chromosomal subregion of interest. Those non-validated non-incorporated terminator nucleotides under polarized FLPs enhance the resolution of the map by a factor of 5.6 and fluorescent light [13]. Swan and colleagues [14] have devel- 17.9, respectively. oped a set of fluorescence polarization-template directed incorporation (FP-TDI) assays for C. elegans. However, this We show the usefulness of this approach by identifying novel approach is labor intensive and requires specialized chemis- alleles of previously characterized genes. In summary, we try and equipment. Using DNA microarrays, large numbers of have taken advantage of a publicly available dataset to adapt reports SNPs can be analyzed in parallel, but the number of individu- a technology widely used for STR analysis to genetic mapping. als that can be analyzed is low because of the high cost per Thanks to the complete automation of genotyping, this chip [15,16]. approach is considerably faster, more reliable and cheaper than previously used mapping strategies in C. elegans or Dro- Besides SNPs, short tandem repeats (STRs) or microsatellites sophila. represent another class of sequence polymorphisms used for genotyping [17-21]. STRs result in fragment length differ- deposited research ences that are either detected on gel-based or capillary Results and discussion sequencers or high-resolution hydrogels (Elchrom Scientific Detection of fragment length polymorphisms (FLPs) Inc.).