SNP HiTLink: a high-throughput linkage analysis system employing dense SNP data
© Fukuda et al; licensee BioMed Central Ltd. 2009
Received: 24 December 2008
Accepted: 24 April 2009
Published: 24 April 2009
During this recent decade, microarray-based single nucleotide polymorphism (SNP) data are becoming more widely used as markers for linkage analysis in the identification of loci for disease-associated genes. Although microarray-based SNP analyses have markedly reduced genotyping time and cost compared with microsatellite-based analyses, applying these enormous data to linkage analysis programs is a time-consuming step, thus, necessitating a high-throughput platform.
We have developed SNP HiTLink (SNP Hi gh T hroughput Link age analysis system). In this system, SNP chip data of the Affymetrix Mapping 100 k/500 k array set and Genome-Wide Human SNP array 5.0/6.0 can be directly imported and passed to parametric or model-free linkage analysis programs; MLINK, Superlink, Merlin and Allegro. Various marker-selecting functions are implemented to avoid the effect of typing-error data, markers in linkage equilibrium or to select informative data.
The results using the 100 k SNP dataset were comparable or even superior to those obtained from analyses using microsatellite markers in terms of LOD scores obtained. General personal computers are sufficient to execute the process, as runtime for whole-genome analysis was less than a few hours. This system can be widely applied to linkage analysis using microarray-based SNP data and with which one can expect high-throughput and reliable linkage analysis.
Recent technological development of high-density SNP chips has made it practical to genotype more than a million SNPs. Because microarray-based dense SNP typing requires less time and typing cost and can provide much more information than PCR-based microsatellite markers, it is now widely recognized as a powerful tool for linkage analysis [1–3]. To apply SNP information to genome-wide high-throughput linkage analysis, however, there are some difficulties as follows. 1) LINKAGE file preparation: Most linkage analysis software accepts LINKAGE format genotype data containing information on each marker for pairwise analysis or that on all markers on each chromosome for multipoint analysis. For example, pairwise analysis of 1000 SNPs on a chromosome using MLINK [4, 5], a pairwise linkage analysis program, means preparing 1000 genotype files and 1000 marker information files, followed by running the program 1000 times. In multipoint analysis, information on the 1000 genotypes or marker information containing intermarker distances should be described in one file. Preparation of these files based on the information contained in the CHP file, which is generated by Affymetrix Genotyping Console ™ from firstly created CEL files in genotyping assays, are laborious and time-consuming for researchers. 2) Typing error: In microarray-based SNP detection, typing error is rare but inevitable because several factors such as the quality of genomic DNA, experimental conditions and the number of samples incorporated in the clustering of genotypes, can lead to inaccurate SNP calling [6–9]. This relatively rare miscalling, however, can lead to critical miscalculation in linkage analysis, particularly when parent genotypes are lacking, or in multipoint analysis. Therefore, estimation and elimination of typing error data would be necessary for reliable results. 3) Linkage disequilibrium (LD) in neighboring markers for multipoint analysis: In algorithms of multipoint linkage analysis, it is usually assumed that all markers are in linkage equilibrium with each other. Markers in LD should be appropriately eliminated to avoid inaccurate calculation, which can be accompanied by inflation of LOD scores [10, 11]. This is particularly important when using recently developed high-density SNP chips.
We have herein developed SNP HiTLink that directly accepts Affymetrix SNP CHP files and perform parametric/nonparametric linkage analyses with quite flexible marker selection functionalities.
SNP HiTLink works under Windows XP SP2 or later/Vista (Use only 32-bit versions of Windows) and unix (supporting perl 5) OS [Additional files 1 and 2]. MLINK (LINKAGE/fastlink), Superlink, Merlin and Allegro should be installed in Unix OS. MLINK is included in FASTLINK package. Allegro is available from deCODE genetics, Inc. At present, SNP HiTLink accepts files in the CHP file format (filename.chp) of the Affymetrix Mapping 100 k/500 k array set and Genome-Wide Human SNP array 5.0/6.0. SNP HiTLink consists of two processes. The first process creates necessary data files by the program described in the Visual Basic programming on Windows OS, and these files are then transferred to Unix OS. The Perl script files invoke necessary linkage programs with necessary data files on Unix OS.
To eliminate markers with typing errors, HWE, call rate, and confidence score are used as the effective indexes because deviations from HWE, lower call rates and higher confidence scores at particular markers sometimes suggest problems with genotyping. 2) To select informative markers useful for linkage analyses, the 'MAF zero test' and 'No call test' will be performed because these markers are totally uninformative. 3) To avoid employing markers in LD in the multipoint analysis, appropriate intermarker distances or D' and r2, which are indexes of LD, can be defined by users.
HWE test: the user sets p-value which is calculated from genotype frequencies in control samples. SNPs with a p-value below the settings are eliminated.
Minimum call rate: the user sets the minimum call rate, which is calculated from "no call/call" ratio in all control samples, to avoid markers with lower call rates suggesting difficulties in genotyping.
MAF zero test: markers where MAFs are zero can be eliminated.
NoCall test (MLINK, Superlink): markers that are not called in any samples analyzed will be eliminated.
Maximum confidence: confidence scores that are reliabilities of signal calling from hybridization can be set here. When the user skips this setting, the default value (for example 0.5 in BRLMM algorithm  as a default) defined in Genotyping Console™, which is Affymetrix genotyping software, will be used.
Interval (Merlin, Allegro): minimum intermarker distances will be set. There are two marker-selecting methods, the min-max method and min MAF and interval method. In the min-max method, the user sets minimum and maximum intervals, then SNP with the highest MAF in the region defined by these intervals will be adopted. On the other hand, the min MAF and interval method select SNPs with MAFs higher than defined, and one SNP locating nearest to the minimum interval from the former SNP will be adopted.
LD: the user sets the maximum D' and r2 scores to eliminate neighboring markers in LD with D' or r2 scores higher than the threshold. The reference LD data file containing all D' and r2 data obtained from the Hapmap database  can be downloaded from our WEB sites. Information of four ethnic populations (CEU, CHB, JPT, and YRI) has been provided as LD data files thus far. Users can make LD data files from their own samples by using LD Data Maker in the Main Menu. Click on LD Data Maker and specify the directory where chip files located.
SNP HiTLink produces a binary file (.lkin file) containing the marker and pedigree information with parameter settings, and this file is transported from Windows OS to Unix OS. Perl programming (run_linkage.pl) performs MLINK, Superlink, Merlin or Allegro against a specified '.lkin' file. Whole genome analysis will be carried out automatically but the user can also specify a chromosome number by option when analyzing only the chromosome of interest. Outputs of haplotype prediction by Allegro in a specific text format are easily visualized on the windows system by using the haplotype viewer implemented in this system. Data are shown in columns and can be copied to an Excel sheet for further use [see also the manual of Additional file 4].
Result and discussion
The runtime for preparing lkin files is less than 10 minutes (usually from about 10 second to a few minutes), and the runtime of whole genome linkage analysis of a pedigree performed using general personal computer was about 4 hours for pairwise analysis, when using all of approximately 1 million markers on Genome-Wide Human SNP array 6.0. For multipoint analysis less than 1 hour was required even in the case of a family including consanguineous loops when intermarker distances were set to be varied from 300 bp to 100 kbp. These results show that extremely dense markers that are now mainly utilized for the genome wide association study (GWAS) can also be utilized for high-throughput linkage analysis.
We have developed the SNP HiTLink, system for executing parametric/nonparametric linkage analysis using SNP data. This is the first and unique system that directly accepts recent 100 K, 500 K and 1 M markers of Affymetrix SNP CHP files and prepares very flexible marker-selecting implementations for linkage analysis, although some convenient pipelines that pass the SNP data to a linkage analysis program [18, 19] or tools for visualization and removal of LD [20, 21] have been developed thus far. The results using this system were comparable or even superior to those obtained using microsatellite markers, convincing us the advantage of using SNP data obtained by DNA microarray for linkage analysis. The number of SNP data located on a single chip is continuing to increase owing to recent developed technologies and demands for dense markers for GWAS. On the other hand, we should be carefully concerned about typing error data when using such dense SNP data for multipoint linkage analysis. Quite flexible marker-selecting implementations on SNP HiTLink will be advantageous from this point of view. Although SNP HiTLink only accepts Affymetrix SNP Chip files, improvements that support multiple platforms for SNP typing such as Illumina are required in the future. Furthermore, more user-friendly interface where analyses can be processed simply (for instance, through integrated single GUI) rather than transporting files from Windows to Unix OS, will be desirable. This system can be widely applied for linkage analysis using microarray-based SNP data, with which one can expect high-throughput and reliable linkage analysis.
The authors would like to thank Drs. Toshihisa Takagi and Tatsuhiko Tsunoda for their insightful comments and helpful suggestions for the system. This work was supported in part by KAKENHI (Grant-in-Aid for Scientific Research) on Priority Areas, Applied Genomics, the 21st Century COE Program, Integrated Database Project, Center for Integrated Brain Medical Science, and Scientific Research (A) from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
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