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Genome Biology 2005, 6:R19 comment reviews reports deposited research refereed research interactions information Open Access 2005Zipperlenet al.Volume 6, Issue 2, Article R19 Method A universal method for automated gene mapping 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. Correspondence: Peder Zipperlen. E-mail: peder.zipperlen@molbio.unizh.ch. Knud Nairz. E-mail: nairz@zool.unizh.ch © 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 InDel sequence polymorphisms.<p>A high-throughput method for genotyping by mapping InDels. This method has been used to create fragment-length polymorphism maps for Drosophila and C. elegans.</p> 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. Background For humans and model organisms, such as worms and flies, the availability of high-density sequence polymorphism maps greatly facilitates the rapid mapping and cloning of genes [1- 3]. Key advantages of most molecular polymorphisms are the fact that they are codominant and in general phenotypically neutral. The vast majority of sequence polymorphisms are single-nucleotide polymorphisms (SNPs). The most direct approach for SNP detection is sequencing of a PCR product spanning the polymorphism, but this is too costly and labor intense for high-throughput genotyping. For this reason, several different strategies and methods have been developed in order to detect SNPs more efficiently. In general, assays can be grouped into strategies, where the nature of the SNP is determined by directly analyzing the primary PCR product and those that require a secondary assay performed on the primary amplification product [4-6]. An important strategy of the first group is the 5' nuclease assay, where allele-specific, dual-labeled fluorescent TaqMan probes guarantee specificity [7]. However, the need for two dual-labeled fluorescent probes, expensive specialized chem- istry and specialized machinery increase the costs per assay of this approach significantly. Similarly, denaturing high-performance liquid chromatography (DHPLC) also analyses the primary amplification product [8]. This approach is based on melting differences of homo- versus heteroduplex DNA fragments under increasingly denaturing conditions and requires no specific labeling of the PCR fragments. However, conditions have to be optimized for every assay, throughput is lim- ited and specialized equipment is required. DHPLC has been used in small-scale genotyping projects in Drosophila melanogaster [9]. Of the methods that detect the SNP in a secondary assay, restriction fragment length polymorphism (RFLP) analysis are very popular [10]. For this purpose, only those SNPs that alter a restriction site are analyzed. A great advantage of Published: 17 January 2005 Genome Biology 2005, 6:R19 Received: 9 September 2004 Revised: 15 November 2004 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 R19.2 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, 6:R19 FLP detection of InDels of various sizes in homozygotes and heterozygotesFigure 1 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. 209 2000 1000 3000 213205201 203 207 211 4000 5000 6000 ZHX-22: 6bp InDel; poly-C stretch (f) 120 125 2000 1000 3000 130 ZH3-23: 1bp InDel; with A addition 1 (d) 2 3R160: 1bp InDel; poly-T stretch 171 174172 173 175 176 6000 12000 8000 4000 2000 177 10000 (e) 1 2 3 EP 171 174172 173 175 176 6000 12000 8000 4000 2000 177 10000 14000 1 2 3 FRT 171 174172 173 175 176 12000 8000 4000 177 16000 20000 41 2 3 FRTEP 209 2000 10000 8000 213205201 203 207 211 4000 6000 209 2000 10000 213205201 203 207 211 4000 8000 6000 120 125 2000 1000 3000 130 4000 5000 1 2 120 125 2000 1000 3000 130 4000 5000 1 2 3 Bristol Bristol Bristol Bristol Hawaii Hawaii Hawaii Hawaii Bristol 117 120118 119 121 122 1600 1200 800 400 2000 123 ZH1-01: 3bp InDel; no A addition (a) 117 120118 119 121 122 4000 8000 2000 6000 10000 123 12000 ZH2-01: 12bp InDel; with A addition 134 136 800 400 1600 150148146144142140138 1200 (b) 174 177175 176 178 179 180 6000 4000 2000 ZH3-05a: 2bp InDel; with A addition (c) 117 120118 119 121 122 4000 3000 2000 1000 5000 123 134 136 100 300 150 [bp] 148146144142140138 200 134 136 400 600 150 [bp] 148146144142140138 200 174 177175 176 178 179 180 4000 3000 2000 1000 Hawaii 174 177175 176 178 179 180 4000 3000 2000 1000 Bristol Hawaii Bristol Bristol Bristol Bristol Hawaii Hawaii Hawaii Hawaii Flourescence Flourescence Fragment length (bp) Fragment length (bp) Fragment length (bp) Fragment length (bp) Fragment length (bp)Fragment length (bp) http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. R19.3 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2005, 6:R19 RFLP analysis is that no specialized equipment is needed and it can be carried out in every laboratory. RFLP maps recently established for Caenorhabditis elegans and Drosophila are used regularly in genotyping projects [2,3,11]. However, RFLP analysis requires significant manual input. Moreover, the use of different restriction enzymes with different reaction requirements adds another level of complexity that makes this method difficult to automate. Primer-extension- based technologies have also gained some prominence [12]. Here, a primer that anneals right next to the polymorphism is extended by one polymorphism-specific terminator nucleotide. Extension products are analyzed by size or, alterna- tively, by differences in the behavior of incorporated versus non-incorporated terminator nucleotides under polarized fluorescent light [13]. Swan and colleagues [14] have developed a set of fluorescence polarization-template directed incorporation (FP-TDI) assays for C. elegans. However, this approach is labor intensive and requires specialized chemis- try and equipment. Using DNA microarrays, large numbers of SNPs can be analyzed in parallel, but the number of individu- als that can be analyzed is low because of the high cost per chip [15,16]. Besides SNPs, short tandem repeats (STRs) or microsatellites represent another class of sequence polymorphisms used for genotyping [17-21]. STRs result in fragment length differences that are either detected on gel-based or capillary sequencers or high-resolution hydrogels (Elchrom Scientific Inc.). One advantage of STRs over SNPs is that they are highly polymorphic and are thus ideal for measuring the degree of variability in natural populations. STRs are, however, present at much lower density than SNPs and are therefore not suita- ble for high-resolution mapping of genes. Interestingly, a significant proportion of the currently available polymorphisms are caused by small insertions or deletions (InDels). Weber et al. [22] identified a genome-wide set of about 2,000 human InDel polymorphisms and estimated that InDels comprise at least 8% and up to 20% of all human polymorphisms. This is in line with the findings of Berger and co-workers [2] who found that 16.2% of polymorphisms in Drosophila are of the InDel type. Also, two independent stud- ies in C. elegans found that InDels constitute between 25% and 28% of all polymorphisms [3,14]. In addition, those stud- ies found that the vast majority of InDels are due to 1-2 base- pair (bp) differences (65% in Drosophila [2], 84% in C. elegans [3]). To take full advantage of this class of small InDel polymorphisms, we have developed a strategy that allows us to detect most, if not all, InDels by analyzing the lengths of primary PCR products on a capillary sequencer at single base-pair resolution. We call these assays fragment length polymorphism (FLP) assays. Importantly, this approach can easily be automated on standard robotic pipetting platforms as it involves a simple PCR reaction setup. Furthermore, allele calling is performed automatically using the Applied Biosystems GeneMa- pper software commonly used for genotyping STRs (Materials and methods). To demonstrate the feasibility of this strategy, we have validated 112 evenly spaced FLP assays at 3 centimorgan (cM) resolution in C. elegans (one every 0.9 megabase-pair (Mbp)) and 54 FLP assays at 4 cM resolution for the Drosophila autosomes. This set of FLP assays allows us to rapidly map mutations to small chromosomal subregions with a minimum of manual input. Furthermore, we provide a list of predicted InDels for which additional assays can be readily designed in the chromosomal subregion of interest. Those non-validated FLPs enhance the resolution of the map by a factor of 5.6 and 17.9, respectively. We show the usefulness of this approach by identifying novel alleles of previously characterized genes. In summary, we have taken advantage of a publicly available dataset to adapt a technology widely used for STR analysis to genetic mapping. Thanks to the complete automation of genotyping, this approach is considerably faster, more reliable and cheaper than previously used mapping strategies in C. elegans or Dro- sophila. Results and discussion Detection of fragment length polymorphisms (FLPs) To detect a FLP, the region of interest is amplified in a standard PCR reaction with one fluorescently labeled primer, and the PCR products are directly analyzed on a capillary sequencer. Fragment sizes are determined automatically rel- ative to an internal size standard with AppliedBiosystem's GeneMapper software (for details see Materials and methods). The software then allocates fragment sizes to previously calibrated genotypes. Taq polymerase has the tendency to catalyze the addition of adenosine (A) to the 3' end of PCR products. This activity could make it difficult to achieve the single base-pair resolution required to assay all available InDels and may hamper allele-calling [23]. However, we have found that the sensitiv- ity of a capillary sequencer and the genotyping software is sufficient to allow for unambiguous allele assignment even for 'difficult' sequences exhibiting 3' A addition. The examples shown in Figure 1a-d illustrate that robust genotyping is fea- sible for 1-bp InDels even when 3' A addition occurs. Another problem is the stuttering of the polymerase when it encoun- ters poly(N) stretches. However, larger InDels are reliably detected by the software in poly(N) stretches (Figure 1f), and in a few difficult cases visual inspection can even resolve and unambiguously assign 'stuttering' 1-bp InDels according to the location and number of peaks (Figure 1e). Genotyping with FLP assays is extremely accurate. In a control experiment, we genotyped all 96 samples of the fly strains R19.4 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, 6:R19 FRT42B and EP0755 for the 1-bp InDel 2R090 and 231 samples homo- and heterozygous for the C. elegans Bristol and Hawaii backgrounds, respectively, for the 1-bp InDel ZH5-16. 2R090 exhibits both stuttering and A addition and hence is especially difficult to resolve (see Additional data file 8). The genotype was correctly and automatically assigned by Gen- eMapper in all 423 assays. Thus, automated genotyping based on FLPs is sensitive down to single base-pair resolution and is extremely robust. The accuracy of FLP mapping is compa- rable to other methods such as TaqMan (error rate less than 1 in 2,000 [24]), minisequencing (99.5% [25]), and pyrosequencing (97.3 % [25]). C. elegans and Drosophila FLP maps In C. elegans, genetic experiments are performed almost exclusively in the background of the standard wild-type strain N2 (C. elegans variety Bristol) [26]. For gene mapping experiments, the polymorphic strain CB4856 (C. elegans, variety Hawaii) has proved extremely useful [3]. When compared to N2, CB4856 contains on average one SNP every 840 bp and approximately 25% of all polymorphisms are InDels [14]. Starting from the dataset previously published by Wicks et al. [3], 112 FLPs that are evenly spaced on the physical map of C. elegans were validated to date (Figure 2a). The confirmation rate of the predicted InDels was 88% (n = 169). Most failures to detect a FLP are probably due to original sequencing errors. The average distance between neighboring FLP assays is about 0.9 Mbp. This physical distance corresponds to about 3 cM, assuming 300 kb per map unit, and encompasses between 100 and a maximum of 500 genes (Figure 2a). The length of the amplicons ranges from 100 to 444 bp, and the fragment length differences are between 1 and 21 bp (Addi- tional data file 9). If necessary, another 2,454 predicted InDels are available to increase the mapping resolution down to 50 kbp on average (Additional data files 12-17). To establish a Drosophila FLP map, a set of 54 FLP assays (12 to 17 per arm of the two major autosomes) was validated from the list of polymorphisms identified by Berger et al. [2] (Fig- ure 2b, and Additional data file 10); high-resolution X-chromosomal SNP and FLP maps have yet to be established. Similarly to C. elegans, the confirmation rate of the predicted Drosophila InDels was 80% (n = 30). Furthermore, another 509 InDels are predicted at 248 sites for which an assay can be established to discriminate between EP and FRT strains (Additional data file 18). The validated Drosophila FLP assays were evenly spaced on the genetic map with an average distance between neighboring assays of about 4 cM, corresponding to an average resolution of 1.77 Mbp on the physical map encompassing 95,55 Mbp [27,28]. Taking into account the non-validated InDels, the maximal average resolution is currently 314 kb or 0.7 cM. On the left arm of chromosome 3, where the genetic map is inexact, FLPs were spaced on the physical map assuming colinearity between the two maps. The length of amplicons ranges from 99 to 365 bp, and the size difference ranges from 1 to 54 bp (Additional data file 9). Our Drosophila FLP assays are in part derived from a set of InDels of size difference 7 bp or more (termed PLPs by Berger et al. [2]). However, since 86.8% of all Drosophila InDels exhibit a length difference of one to six nucleotides [2], so far only a small subset of the available InDels has been covered. The approach presented here significantly increases the number of possible FLP assays for genotyping and offers a greater flexibility and higher resolution. FLP mapping of C. elegans genes To demonstrate the usefulness of the C. elegans FLP map, we mapped three previously characterized mutations on chromosome II that exhibit diverse phenotypes. Those were the centrally located let-23(sy1) allele that causes an 80% penetrant vulvaless phenotype [29], rol-1(e91) in the middle of the left chromosome arm, which causes the animals to roll around their body axis [30], and the unc-52(e444) mutation located at the right end of the chromosome, which results in a paralyzed phenotype [31]. Mutant hermaphrodites were crossed with CB4856 males, and wild-type F 1 cross-progeny was selected (F 1 self-progeny would exhibit a mutant phenotype). Finally, mutant self-progeny was isolated in the F 2 generation and used for genotyping (Figure 3a). To minimize the number of PCR reactions, we pursued a two-step strategy. First, we determined chromosomal linkage by analyzing 16 individual F 2 animals (corresponding to 32 chromosomes in total) with one centrally located FLP assay per chromosome (Tier 1, Figure 2a). This allowed us to establish clear linkage to chromosome 2 for all three mutations (Additional data file 2). Surprisingly, the rol-1(e91) mutation showed linkage to the X chromosome of N2 in addition to chromosome II. This pseudo-linkage could be due to a suppressor of the Rol phenotype present on the CB4856 X chromosome. In a second step, 48 F 2 animals for each mutation were analyzed with eight FLP assays along chromosome 2 (Tier 2, Figure 2a). In C. elegans and Drosophila FLP mapsFigure 2 (see following page) C. elegans and Drosophila FLP maps. (a) The C. elegans FLP map. Marker names comprise a ZH prefix followed by the chromosome number and a unique identifier number. Markers used in first-level assays (Tier 1) for determination of chromosomal linkage are in red, those used for second-level assays (Tier 2) for higher resolution mapping are in black. (b) The Drosophila FLP map of chromosomes 2 and 3. The FRT sites and EP elements are symbolized by blue and green triangles, respectively. The strains that were genotyped are shown below each chromosome. Green indicates the EP genotype, blue the FRT genotypes and new alleles are shown in other colors. http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. R19.5 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2005, 6:R19 Figure 2 (see legend on previous page) I ZH1-16 ZH1-17 ZH1-10a ZH1-25 ZH1-07 ZH1-03 ZH1-21 ZH1-01 ZH1-22 ZH1-23 ZH1-15 ZH1-05 ZH1-08 ZH1-09 ZH1-06 ZH1-24 0 5 10 15 20 M b ZH2-15 ZH2-04a ZH2-05 ZH2-16 ZH2-06a ZH2-07 ZH2-17 ZH2-13 ZH2-19 ZH2-01 ZH2-02 ZH2-20 ZH2-09 ZH2-10 ZH2-11 ZH2-12 ZH2-23 ZH3-06 ZH3-08 ZH3-15 ZH3-04 ZH3-02 ZH3-05a ZH3-10a ZH3-23 ZH3-11 ZH3-12 ZH3-13 ZH3-07 ZH4-04a ZH4-05 ZH4-06 ZH4-07 ZH4-16 ZH4-08 ZH4-02 ZH4-03 ZH4-17 ZH4-18 ZH4-09 ZH4-19 ZH4-20 ZH4-10a ZH4-21 ZH4-11 ZH4-12 ZH4-22 ZHX-16 ZHX-17 ZHX-03 ZHX-08 ZHX-13 ZHX-15 ZHX-10 ZHX-02 ZHX-12 ZHX-07 ZHX-11 ZHX-05 ZHX-06 ZHX-22 ZHX-23 ZH5-02a ZH5-13 ZH5-03a ZH5-12 ZH5-11 ZH5-06 ZH5-18 ZH5-17 ZH5-01 ZH5-16 ZH5-05 ZH5-15 ZH5-04 ZH5-14 ZH5-22 ZH5-09 ZH5-21 ZH5-08 ZH5-20 ( a) ( b) EP2L FRT2L EP2R FRT2R FRT40A,w + , cl 2L017 2L030 2L038 2L051 2L057 2L069 2L075 2L088 2L090 2L093 2L119 2L143 5 2017.512.5 15107.52.5 EP0511 FRT40A EP2R FRT2R FRT2L EP2L FRT42D,w + , cl 2.5 12.5107.55 2017.515 FRT42D EP0755 2R017 2R118 2R109 2R060 2R083 2R068 2R051 2R039 2R096 2R130 2R139 2R124 EP3L FRT3L EP3R FRT3R FRT80A,w + , cl 3L021 3L031 3L127 3L041 3L058 3L064 3L076 3L083 3L086 3L105 3L148 3L094 EP3104 FRT80A 5 22.52017.512.5 15107.52.5 EP3R FRT3R FRT3L EP3L FRT82,w + , c l EP0381FRT82B 3R061 3R192 3R186 3R160 3R151 3R092 3R122 3R074 3R169 3R221 3R224 3R204 2522.52017.52.5 12.5107.55 15 27.5 II III I V X V L G CEN CEN 2 L 2R 3 L 3R yw(WG) yw(WG) yw(GT1) yw(GT1) yw(WG) yw(GT1) yw(GT1) yw(WG) EP FRT Novel alleles No amplification M b M b ZH1-18a ZH1-27 ZH1-34 ZH2-25 ZH2-27 ZH2-28 ZH3-17a ZH3-25 ZH3-26 ZH3-28 ZH3-32 ZH3-35 ZH5-23 ZHX-24 ZHX-21a Assays used for chromosomal linkage (tier 1) R19.6 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, 6:R19 this way, we could narrow down the three mutations to the correct chromosomal subregions (Additional data files 3-5). We used the same strategy to map the zh41 mutation that was identified in a forward genetic screen for mutants exhibiting a loss of egl-17::gfp expression in the vulval cell linage ([32] and I. Rimann and A. Hajnal, unpublished work). Analysis with Tier 1 established linkage to chromosome 1 (Figure 3b), and Tier 2 narrowed down the candidate region to an interval of 2.2 Mbp containing 498 genes (Figure 3c). The phenotype of zh41 animals is similar to the phenotype caused by loss-of- function mutations in lin-11, which maps to the same interval in the center of chromosome I [33]. Like lin-11 mutants, zh41 animals exhibit a penetrant protruding vulva (Pvl) phenotype, and staining of the adherens junctions with the MH27 antibody showed defects in the formation of the vulval torroid rings (Figure 3d) [33]. Subsequent sequencing of the lin-11 locus in zh41 animals revealed a point mutation that results in a change of leucine 274 to phenylalanine. Furthermore, zh41 failed to complement lin-11(n389), indicating that the zh41 mutation in the lin-11 open reading frame (ORF) is responsi- ble for the vulval phenotype. In cases where a mutation maps to an interval that contains no obvious candidate gene, we first screen for additional informative recombinants by FLP analysis and then refine the map position by extracting more FLPs from our set of non- validated InDels (Additional data files 12-17) and by genotyping existing SNPs in the candidate interval [3]. In many cases, this resolution is sufficient to identify the affected gene through RNA interference (RNAi) analysis of the genes in the corresponding interval [34]. (See Additional data file 6 for a detailed flowchart of the mapping process). In summary, FLP mapping in C. elegans allows us to rapidly map a mutation down to a small region containing, on average, 200 candidate genes by crossing a mutant strain to CB4856 and analyzing 48 F 2 animals with 300 to 500 PCR reactions. Genotyping Drosophila strains with FLP assays In contrast to the well defined genetic backgrounds used for C. elegans, zebrafish (Danio rerio) or Arabidopsis genetics, Drosophila strains are very heterogeneous and of ill-defined origin [2,9,11]. In this respect, gene mapping in Drosophila resembles human genetics in that standard inbred lines do not exist and the genotypes of the parental lines have to be determined first. As genome-wide polymorphism databases for reference strains are available [2,11], a line of interest can be crossed with two reference strains, such as EP and FRT (see below). Owing to the codominant character of sequence polymorphisms, at least one of the two respective crosses will distinguish between the mutant and the mapping chromosomes. To further facilitate mapping with our set of FLP assays, we genotyped several common laboratory lines such as two 'wild-type' yw strains for the whole set, four FRT- Minute or FRT-cell-lethal strains at the relevant autosomal arms [35], as well as the FRT and EP reference strains at both relevant autosomal arms (Figure 2b). Surprisingly, the FRT and EP lines are largely not of FRT or EP genotype on the chromosome arm for which they have not been calibrated. Overall, we found novel alleles for 18 of the 48 assays, and in an extreme case, we even observed five different alleles in five examined strains (2R017, Figure 2b). This result further high- lights the heterogeneity of Drosophila strains (see Additional data file 1 for further details on FLP calibration and fly genetics). FLP mapping in Drosophila In a genetic screen devised to isolate genes that regulate growth and are situated on chromosome 2R, we found a complementation group characterized by a mild overgrowth phenotype (Figure 4b (2), and C. Rottig and E.H., unpublished work). From a cross between allele VI.29 and EP0755 we recovered three types of recombinant chromosomes: recombinants with a crossover proximal or distal to the mutation, respectively, and double-crossovers (Figure 4a, see also Additional data file 1 for further details on the crossing scheme). The mutation could be placed 16.9 cM proximal to EP0755 and 38.7 cM distal to FRT42D. The FLPs in the recombinant flies were directly analyzed without backcross- ing the recombinant chromosome into a parental strain background. DNA was prepared from recombinants by a novel high-throughput protocol (see Materials and methods). We genotyped 34 distal crossover events, 40 proximal crossovers, and eight double-crossovers. This analysis placed the mutation between markers 2R096 and 2R109 (Figure 4c). This interval includes the tumor suppressor hippo [36], and subsequent complementation analysis confirmed VI.29 as a weak hippo allele (data not shown). Furthermore, data from this and other FLP mappings in this region allowed us to further refine the genetic map (Additional data file 11). This kind of experimental data is helpful to space new FLP assays more evenly on the genetic map should the available map turn out to be inexact. If the resolution of the validated FLP map is too low to identify a candidate gene, we further refine the map position by several approaches. First, we design novel FLP-assays in the region of interest and genotype the most informative recombinants from the first round of FLP mapping (Additional data file 18). Second, we genotype recombinants with SNPs available in the region of interest and resolve them by RFLP, sequencing or DHPLC [2,9]. Third, we perform complementation analysis with recently established Drosophila lines with molecularly defined deletions [37,38]. (See Additional data file 7 for a detailed flowchart illustrating the mapping process.) Conclusions We have developed an automated method to detect most nat- urally occurring InDel polymorphisms at single base-pair res- http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. R19.7 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2005, 6:R19 olution. Since a significant fraction of polymorphisms are caused by InDels of only a few base pairs (for example, 8% to 20% in humans [22]) the resolution of the medium-density FLP maps can be greatly increased where necessary, for example during the positional cloning of genes. We are therefore continually designing new FLP assays according to our specific needs using the predicted FLPs (Additional data files 12-18). The full automation of the genotyping has three main advantages when compared to manual methods. First, the error rate (the number of wrongly assigned genotypes) is extremely low, as it was not measurable in 432 assays. Sec- ond, genotyping can be done very rapidly and at a high- throughput with little manpower. The automatic allele-calling, in particular, saves much time. As the identification of informative recombinants is usually the rate-limiting step, FLP mapping is very helpful in extracting the few relevant recombinants from a large number of samples. Third, thanks to the standardized conditions, the low error rate and the absence of a secondary assay, FLP mapping is considerably cheaper than the previously published 'manual' mapping methods [2,3]. Unlike other high-throughput methods like TaqMan, Pyrosequencing, DHPLC, fluorescence polarization or primer-extension assays, FLP mapping does not require any investment in specialized equipment. It can be done in any molecular biology lab with access to a sequencing facility equipped with a capillary- or gel-based system, which usually includes the genotyping software. PCR costs are marginally FLP mapping in C. elegansFigure 3 FLP mapping in C. elegans. (a) Crossing scheme used to map mutations generated in the N2 Bristol background. The different classes of recombinants recovered in the F 2 generation are shown. (b) Analysis of the zh41 mutation with Tier 1 assays establishes linkage to chromosome I. (c) Analysis with Tier 2 places zh41 between assays ZH1-01 and ZH1-15. ND, no data as a result of PCR reaction failure. (d) Ventral views of the vulva in wild-type and zh41 L4 larvae stained with the adherens junction antibody MH27 [44]. In the wild type, the vulval cells have fused to generate the torroids that appear as concentric rings. zh41 mutants exhibit the same fusion defects observed in other lin-11 alleles [33]. 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ND ND P 0 F 1 F 2 CB4856 (Hawaii) m* m* x m* Isolation of wild-type cross-progeny Isolation of mutant self-progeny N2 (Bristol) (a) m* m* Crossover to right of mutation Crossover to left of mutation m* m* m* m* Crossovers to right and left of mutation wild-type zh 41 (d)(b) (c) 0% 20% 40% 60% 80% 100% 12345X Chromosome % Bristol 1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 Lysate ZH1-10a ZH1-07 ZH1-03 ZH1-01 ZH1-15 ZH1-05 ZH1-08 ZH1-06 T i er 2 zh 41 subchromosomal region T i er 1 zh 41 chromosomal linkage Recombinanats Informative R19.8 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, 6:R19 Figure 4 (see legend on next page) hp o 42-20 hp o V I.29 (a) (b) (c) × × Isolation of EP/FRT virgins EP m - cl* m - FRT Balancer m - cl* M + cl* Crossover distal to mutation Crossover proximal to mutation M + cl* Isolation of white-eyed wild-type mosaics Isolation of red-eyed mutant mosaics CEN CEN CEN CEN CEN CEN CEN Crossovers distal and proximal to mutation Isolation of red-eyed wild-type mosaics 1 y w 3 2 Double crossover R1 FRT FRT FRT ND FRT FRT FRT EP EP EP EP EP FRT Proximal crossover R1 FRT FRT FRT FRT EP EP EP ND ND ND ND ND ND R2 FRT FRT FRT EP EP EP EP EP EP FRT FRT FRT FRT R2 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND R5 FRT FRT FRT EP EP EP EP EP EP FRT FRT FRT FRT R3 FRT FRT FRT ND FRT EP EP ND ND ND ND ND ND R7 FRT FRT EP ND EP EP EP EP EP EP FRT FRT FRT R4 FRT EP EP ND EP ND EP ND ND ND ND ND ND R8 EP EP EP ND EP EP EP EP EP EP EP EP FRT R5 FRT FRT FRT ND FRT EP EP ND ND ND ND ND ND R11 FRT FRT FRT ND FRT FRT FRT FRT EP EP EP EP FRT R6 FRT FRT FRT ND FRT FRT EP ND ND ND ND ND ND R12 EP EP EP ND EP EP EP EP EP EP FRT FRT FRT R7 FRT EP EP ND EP EP EP ND ND ND ND ND ND R13 FRT FRT EP ND EP EP EP EP EP EP FRT FRT FRT R8 EP EP EP ND EP EP EP ND ND ND ND ND ND R9 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND Distal crossover R2 ND ND ND FRT ND ND FRT FRT ND FRT FRT FRT FRT R10 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND R3 ND ND ND ND FRT ND FRT FRT FRT FRT FRT EP EP R11 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND R4 ND ND ND ND ND ND FRT FRT FRT ND FRT ND FRT R12 FRT FRT EP ND EP EP EP ND ND ND ND ND ND R5 ND ND ND ND ND ND FRT FRT FRT FRT FRT EP EP R13 FRT FRT FRT ND FRT FRT FRT FRT EP ND ND ND ND R6 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT EP R14 FRT EP EP ND EP EP EP ND ND ND ND ND ND R7 ND ND ND ND ND ND FRT FRT FRT FRT EP EP EP R15 FRT FRT FRT ND FRT EP EP ND ND ND ND ND ND R8 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT EP R16 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND R9 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT FRT R17 FRT FRT FRT ND FRT EP EP ND ND ND ND ND ND R10 ND ND ND ND ND ND FRT FRT EP EP EP EP EP R18 FRT FRT FRT ND FRT FRT FRT FRT EP ND ND ND ND R11 ND ND ND ND ND ND FRT FRT FRT FRT EP EP EP R19 FRT FRT EP ND ND EP EP ND ND ND ND ND ND R12 ND ND ND ND ND ND FRT FRT EP EP EP EP EP R20 FRT FRT FRT EP EP ND EP ND ND ND ND ND ND R13 ND ND ND ND ND ND FRT FRT FRT FRT FRT ND FRT R21 EP EP EP ND EP EP EP ND ND ND ND ND ND R14 ND ND ND ND ND ND FRT FRT FRT EP EP ND EP R22 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND R15 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT EP R23 FRT FRT FRT ND FRT FRT EP ND ND ND ND ND ND R16 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT EP R24 FRT FRT FRT FRT EP EP EP ND ND ND ND ND ND R17 ND ND ND ND ND ND FRT FRT FRT ND FRT EP EP R25 FRT FRT EP ND EP EP EP ND ND ND ND ND ND R18 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT FRT R26 FRT FRT FRT FRT EP EP EP ND ND ND ND ND ND R19 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT EP R27 FRT FRT FRT ND FRT FRT FRT FRT EP ND ND ND ND R20 ND ND ND ND ND ND FRT FRT FRT ND FRT EP ND R28 FRT FRT FRT FRT EP EP EP ND ND ND ND ND ND R21 ND ND ND ND ND ND FRT FRT FRT FRT ND FRT FRT R29 FRT FRT FRT EP EP EP EP ND ND ND ND ND ND R22 ND ND ND ND ND ND FRT FRT FRT EP ND EP EP R30 FRT FRT FRT ND FRT EP EP ND ND ND ND ND ND R23 ND ND ND ND ND ND FRT FRT FRT FRT EP EP EP R31 FRT FRT FRT ND ND FRT FRT FRT EP ND ND ND ND R24 ND ND ND ND ND ND FRT FRT FRT EP EP EP EP R32 FRT EP EP ND EP EP EP ND ND ND ND ND ND R25 ND ND ND ND ND ND FRT FRT FRT EP EP EP EP R26 ND ND FRT ND ND ND FRT FRT FRT EP EP EP EP More recs R10 EP EP EP ND EP EP EP EP EP EP EP EP EP R27 ND ND ND ND ND ND FRT FRT FRT FRT FRT EP EP R14 FRT FRT EP ND EP EP EP EP EP EP EP EP EP R28 ND ND ND ND ND ND FRT FRT FRT FRT EP EP EP R15 FRT FRT FRT ND FRT FRT FRT EP EP EP EP EP EP R29 ND ND ND ND ND ND FRT FRT FRT FRT FRT FRT FRT R16 FRT FRT FRT EP EP EP EP EP EP EP EP EP EP R30 ND ND ND ND ND ND FRT FRT FRT FRT FRT EP EP R3 FRT EP EP ND EP EP EP EP EP EP EP EP EP 29 1 FRT FRT FRT ND FRT FRT FRT FRT ND FRT ND FRT FRT R4 FRT FRT FRT ND FRT EP EP EP EP EP EP EP EP 29 2 FRT FRT FRT ND FRT FRT FRT FRT ND FRT FRT EP EP R6 FRT EP EP ND EP EP EP EP EP EP EP EP EP 29 3 FRT FRT FRT ND FRT FRT FRT FRT ND FRT EP EP EP R9 EP EP EP ND EP EP EP EP EP EP EP EP EP 29 4 FRT FRT FRT ND FRT FRT FRT FRT FRT FRT ND FRT EP 29 5 FRT FRT FRT ND FRT FRT FRT FRT FRT EP EP EP EP 29 8 ND ND ND ND ND ND ND ND FRT EP EP ND ND 2R017 2R039 2R051 2R060 2R068 2R083 2R090 2R096 2R109 2R118 2R124 2R130 2R139 2R017 2R039 2R051 2R060 2R068 2R083 2R090 2R096 2R109 2R118 2R124 2R130 2R139 Cross Recombinant number Cross Recombinant number F 0 F 1 F 2 http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. R19.9 comment reviews reports refereed researchdeposited research interactions information Genome Biology 2005, 6:R19 higher because of the use of fluorescently labeled primers, but there are no added expenses for secondary enzymatic assays. It seems likely that in most organisms the frequency of polymorphisms caused by InDels is in the same range as found in humans, C. elegans or Drosophila. For example, 7.3% of the Arabidopsis sequence polymorphisms are InDels [39]. Thus, FLP mapping can easily be adapted to any organism for which polymorphism maps have been established, as there is no conceptual difference between human, Arabidopsis, C. elegans or Drosophila FLPs. Materials and methods C. elegans and Drosophila culture techniques and alleles Culturing and crossing of C. elegans was done according to standard procedures described in [26]. C. elegans alleles used were: LG I: lin-11(zh41), lin-11(n389); LG II: rol-1(e91), let- 23(sy1), unc-52(e444). Drosophila strains and the genetic screen have been described previously [9,35,40-42]. Single worm DNA extraction Adult worms were collected in 10 µl lysis buffer (50 mM KCl, 10 mM Tris pH 8.2, 2.5 mM MgCl 2 , 0.45% NP-40, 0.45% Tween-20, 100 µg/ml freshly added proteinase K) and incubated for 60 min at 65°C followed by heat-inactivation of proteinase K at 95°C for 10 min. Before PCR, 90 µl double- distilled H 2 O (ddH 2 O) was added to obtain a total volume of 100 µl per lysate. Fly DNA extraction DNA from recombinant flies was extracted in bulk by squishing flies through mechanical force in a vibration mill (Retsch MM30) programmed to shake for 20 sec at 20 strokes per second [43]. Single flies were placed into wells of a 96-well for- mat deep-well plate with each well filled with 200 µl squishing buffer (10 mM Tris-Cl pH 8.2, 1 mM EDTA, 0.2% Triton X-100, 25 mM NaCl, 200 µg/ml freshly added proteinase K) and a tungsten carbide bead (Qiagen). The deep-well plate was then sealed with a rubber mat (Eppendorf) and clamped into the vibration mill. (Tungsten carbide beads can be recycled: after an overnight incubation in 0.1 M HCl and thorough washing in ddH 2 O the beads are virtually free of contaminating DNA.) Debris was allowed to settle for about 5 min, and 50 µl of each supernatant were transferred into a 96- well PCR plate. The reactions were incubated in a thermo- cycler for 30 min at 37°C and finally for 10 min at 95°C to heat-inactivate proteinase K. Before PCR amplification, the crude DNA extracts were diluted 20-fold to reduce the con- centration of proteins that might be harmful for the capillary sequencer. PCR and FLP fragment analysis Diluted single-worm lysates (2 µl samples) or single fly extracts were added to 23 µl PCR reaction mix. Final concen- trations in the PCR reaction were: 0.4 µM forward/reverse primer, 0.2 mM dNTPs, 2 mM MgCl 2 , 1x PCR reaction buffer, 0.25 U EuroTaq polymerase (Euroclone). PCR reaction setup was done in 96-well plates using a Tecan Genesis pipetting robot with disposable tips. PCR was carried out in two MJR thermo-cyclers that are integrated into the robot. The current setup allows for the sequential processing of six 96-well plates at a time. Cycling parameters were 2 min 95°C, 20 sec 95°C, 20 sec 61°C (-0.5°C for each cycle), 45 sec 72°C (for 10 cycles) followed by 24 cycles of 20 sec 95°C, 20 sec 56°C, 45 sec 72°C and a 10 min 72°C final extension. Following PCR, reactions were diluted 1:100 in water, and 2 µl diluted PCR products were mixed with 10 µl HiDi formamide containing 0.025 µl LIZ500 size standard (Applied Biosystems). This dilution before analysis on the capillary sequencer is necessary to reduce signal intensity because too strong signals compromise data analysis. In addition, sample dilution reduces the risk of damaging the capillaries with proteins or lipids present in the crude lysates. The dilution was done with standard tips using the Tecan Genesis pipetting station. Car- ryover of fragments was prohibited by a simple wash step with H 2 O. Fragments were analyzed on an ABI3730 capillary sequencer using POP7 polymer according to standard procedures. Data were analyzed using AppliedBiosystems GeneM- apper software and raw data were treated further with Microsoft Excel. Additional data files The following additional data are available with the online version of this article. Additional data file 1 contains general information on fly genetics. Further C. elegans mapping results are given in Additional data files 2,3,4 and 5. Detailed flowcharts illustrating the FLP mapping process are shown in Additional data files 6 and 7. Additional data file 8 contains electropherograms demonstrating the accuracy of allele-calling. Additional data files 9 and 10 contain tables of primer and sequence data of experimentally verified FLP assays in C. elegans and Drosophila, respectively. Additional data file 11 contains a table of the refined genetic distances for FLP assays on the right arm of Drosophila chromosome 2. Additional non-validated FLPs FLP mapping in DrosophilaFigure 4 (see previous page) FLP mapping in Drosophila. (a) Crossing scheme used to map mutations generated in the FRT background and recombined with an EP line. The different classes of recombinants recovered in the F 2 generation are shown. (b) Big head phenotypes of the hippo null allele hpo 42-20 (1) and the VI.29 mutation (2). A wild-type control is shown in (3). (c) FLP mapping of the VI.29 mutation on chromosome 2R. Analysis of the different classes of recombinants places the mutation between markers 2R096 and 2R109 (dashed red line). Informative recombinants are boxed in red. ND, not determined or no data as a result of PCR reaction failure. R19.10 Genome Biology 2005, Volume 6, Issue 2, Article R19 Zipperlen et al. http://genomebiology.com/2005/6/2/R19 Genome Biology 2005, 6:R19 can be found in Additional data files 12,13,14,15,16 and 17 (C. elegans) and Additional data file 18 (Drosophila). Additional data file 1General information on fly geneticsGeneral information on fly geneticsClick here for additional data fileAdditional data file 2Further C. elegans mapping resultsFurther C. elegans mapping resultsClick here for additional data fileAdditional data file 3Further C. elegans mapping resultsFurther C. elegans mapping resultsClick here for additional data fileAdditional data file 4Further C. elegans mapping resultsFurther C. elegans mapping resultsClick here for additional data fileAdditional data file 5Further C. elegans mapping resultsFurther C. elegans mapping resultsClick here for additional data fileAdditional data file 6Detailed flowcharts illustrating the FLP mapping processDetailed flowcharts illustrating the FLP mapping processClick here for additional data fileAdditional data file 7Detailed flowcharts illustrating the FLP mapping processDetailed flowcharts illustrating the FLP mapping processClick here for additional data fileAdditional data file 8Electropherograms demonstrating the accuracy of allele-callingElectropherograms demonstrating the accuracy of allele-callingClick here for additional data fileAdditional data file 9Tables of primer and sequence data of experimentally verified FLP assays in C. elegansTables of primer and sequence data of experimentally verified FLP assays in C. elegansClick here for additional data fileAdditional data file 10Tables of primer and sequence data of experimentally verified FLP assays in DrosophilaTables of primer and sequence data of experimentally verified FLP assays in DrosophilaClick here for additional data fileAdditional data file 11A table of the refined genetic distances for FLP assays on the right arm of Drosophila chromosome 2A table of the refined genetic distances for FLP assays on the right arm of Drosophila chromosome 2Click here for additional data fileAdditional data file 12Additional non-validated FLPs (C. elegans)Additional non-validated FLPs (C. elegans)Click here for additional data fileAdditional data file 13Additional non-validated FLPs (C. elegans)Additional non-validated FLPs (C. elegans)Click here for additional data fileAdditional data file 14Additional non-validated FLPs (C. elegans)Additional non-validated FLPs (C. elegans)Click here for additional data fileAdditional data file 15Additional non-validated FLPs (C. elegans)Additional non-validated FLPs (C. elegans)Click here for additional data fileAdditional data file 16Additional non-validated FLPs (C. elegans)Additional non-validated FLPs (C. elegans)Click here for additional data fileAdditional data file 17Additional non-validated FLPs (C. elegans)Additional non-validated FLPs (C. elegans)Click here for additional data fileAdditional data file 18Additional non-validated FLPs (Drosophila)Additional non-validated FLPs (Drosophila)Click here for additional data file Acknowledgements We are grateful to Carmen Rottig for providing us with the novel hippo mutant and to DJ Pan for the hpo 42-20 mutation. 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In summary, we have taken advantage of a publicly available dataset to adapt a technology widely used for STR analysis to genetic. is performed automatically using the Applied Biosystems GeneMa- pper software commonly used for genotyping STRs (Materials and methods). To demonstrate the feasibility of this strategy, we have. cloning of genes [1- 3]. Key advantages of most molecular polymorphisms are the fact that they are codominant and in general phenotypically neutral. The vast majority of sequence polymorphisms

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