Microarray Analysis: Genome-wide Association

Table of Contents

Letter from the Editor
Index of Experts
Q1: How do you determine the number and type of SNPs to interrogate?
Q2: What is your process for choosing the necessary sample size for a study?
Q3: What are your requirements in selecting an appropriate control group?
Q4: How do you correct p-values for multiple hypothesis testing?
Q5: How do you establish thresholds to declare statistically significant association?
Q6: What methods do you use to distinguish between false positives and true positives or noise?
List of Resources

Download the PDF version here

Letter from the Editor

Welcome to the latest issue of Genome Technology's technical reference guide that focuses, once again, on microarrays—this time with an eye toward genome-wide association studies. Previously, GT brought you tips on sample preparation, quality control and confirmation and, most recently, the specifics of cDNA microarrays. Now, we continue to address the questions that arise in the microarray field by presenting a technical guide that shows you strategies to analyze the data from those well-prepared, high-quality, experiment-specific microarray experiments that you've run.

In the end, even if you've been über-careful about your sample preparation or normalization steps, it is how you analyze your data that really matters. A slip-up here or there and you've dug yourself into a false-positive hole. So we've rounded up a crew of experts to give their advice on how to choose your sample size and correct your p-values as well as how to distinguish true positive associations from all that noise. Without further ado, we'll let you jump right into the nitty-gritty on how to deal with analyzing all that microarray data.

— Ciara Curtin

Index of Experts

Genome Technology would like to thank the following contributors for taking the time to respond to the questions in this tech guide.

Eleazar Eskin
Assistant Professor
University of California, Los Angeles

Tom LaFramboise
Assistant Professor
Case Western Reserve University

Jae Lee
Associate Professor
University of Virginia School of Medicine

Constantin Polychronakos
Professor
McGill University Health Center

George Uhl
Chief, Neurobiology Branch NIH IRP, DHSS
Associate Professor
Johns Hopkins University

Jean Claude Zenklusen
Staff Scientist
National Cancer Institute, NIH

Q1: How do you determine the number and type of SNPs to interrogate?

When performing association studies, the first step of the study is the design phase where you decide the region of the genome in which to perform the association study. If you are interested in a specific region such as a single candidate gene, then it makes sense to choose a smaller number of SNPs. Due to the correlation structure of the genome, not all SNPs in the region must be genotyped, but only a set of tag SNPs or a subset of the SNPs that serve as markers for the remaining SNPs. Many tools are available to select tag SNPs. One of the most popular is Tagger.

— Eleazar Eskin

Given the explosion in the number of SNPs available on the commercial arrays, there is generally no need to select SNPs to interrogate. The two products that we are most familiar with can currently interrogate between 500,000 and 1 million SNPs. This level of resolution captures most of the single nucleotide-level germ line variation in the human genome (especially for non-African populations), and is sufficient to detect most somatic copy number changes in cancer cells. One application for which the resolution may be insufficient is the detection of germ line copy number variants, which tend to be smaller than those that occur somatically. Some CNVs are detectable at this resolution. However, if the researcher is interested in interrogating samples for a specific CNV, it may be best to design a custom array based upon known SNPs in the region of interest.

— Tom LaFramboise

A bigger number of SNPs (consequently a denser SNP-map resolution) is generally beneficial for accurately identifying the chromosome locations that are responsible for disease phenotypes. Thus, the decision on the number and type of SNPs can be made based both on the desired resolution of the SNP map and a budgetary consideration under a linkage association study. The other important parameter will be the sample size of different biological subjects or patients for SNP screening which can be derived as a mathematical function of the heritability, proportion of genetic variability compared to total variability of disease phenotype. The higher the heritability, the smaller sample size is required for the desired statistical power.

— Jae Lee

Number: The short answer is: the more the better; however, financial and logistical constraints always dictate a compromise. For technologies that can pick specific SNPs to tag haplotypes, additional coverage per additional SNP rapidly declines somewhere between 500,000 and 1 million. With technologies that must rely on random selection of SNPs, multiply these numbers by a factor of 1.5 to 2. These numbers also increase for subjects of African ancestry, as these individuals are known to have shorter LD blocks and will need additional markers for coverage. Type: Assuming that the technology allows the choice, the priority to which most markers should be allocated is to tag LD blocks as thoroughly as possible. Selection of tags for HapMap SNPs is straightforward. Tagging at an r2 cutoff of 0.8 means that for any given HapMap SNP, the array contains at least one marker that will provide more than 80 percent of the information that would be obtained by genotyping the SNP in question. Large LD blocks ontaining many known SNPs will, in most cases, require tagging a broad enough spectrum of allele frequencies to tag, reasonably well, most unknown or untyped variants. This may not be the case in smaller blocks. It should also be kept in mind that HapMap r2 values, based on 60 genotypes (120 chromosomes) may have large confidence intervals for variants with low allele frequencies, resulting in tagging lower than that intended by the array designers. In addition to an effort to tag all SNPs in each block, chip design should maximize the detection of copy-number variants. Some CNVs are well tagged by haplotypes of adjacent SNPs but some may require detection by quantitative assessment of hybridization to sequences that need not be polymorphic.

— Constantin Polychronakos

There is increasing evidence that the fine structures of linkage disequilibrium can vary subtly from population to population in ways that are likely to alter the information achieved by any set of Tag SNPs. We have thus continued to approach GWA by genotyping as many SNPs as are feasible, creating densities of information that sample more and more of the genomic diversity as more and more SNPs are available. Such an approach is tempered by the modest amounts of information (and possible additions to noise) that SNPs with very low minor allele frequencies provide (especially when seeking common allelic variants for common disorders). We thus typically remove data from SNPs with minor allele frequencies less than 2 percent from data derived from commercial microarray sets.

— George Uhl

We do not select the SNPs to be used, since the format that we employ is commercially available. This helps in avoiding all the problems with assay optimization since the content in the chips is selected to have a narrow range of hybridization properties, thus simplifying the work. Regarding the number of SNPs to use (type of chip, really), it depends on the use. If we are employing them to track purity or identity of long-term cultures [quality control], we use the simpler, cheaper, 10K arrays that have more than enough points to make a clear identification. For regular discovery projects we use the 100K format for several reasons.

Legacy: Our largest project covers 1,000 to 1,500 samples collected through five years. For consistency's sake, we kept using the same platform.

Internal quality checks: Being able to perform the hybridization in two separate chips with the same sample allows for independent analysis of the results, and the subsequent cross-check increases the confidence in concordant data.

Range of amplicons: Large range of amplicons in 100K allows for better representation of genomic regions.

For Formalin-Fixed Paraffin-Embedded (FFPE) analysis, we use the 250K Sty chip since the lower range of amplicons is best suited for the degraded DNA obtained from such samples.

—Jean Claude Zenklusen

Q2: What is your process for choosing the necessary sample size for a study?

There are several tools available for determining the sample size. The choice is determined by the desired statistical power of the association study. The statistical power of a study depends on both the strength of the effect and the sample size. Once the set of SNPs is chosen, we must make assumptions about the strength of the effect that we hope to discover. The strength of the effect is usually parameterized in terms of relative risk which is an individual's increase in probability of having the disease if an individual has the causal variant. A popular tool for computing the sample size is CaTS.

— Eleazar Eskin

Of course, the short answer is: "as large a sample as possible." Given the difficulty of collecting samples and the cost of running arrays, the practical truth is that most researchers make the best of the samples they have available. This set of available samples can be augmented by publicly available downloadable data. Such posted data is the most useful when the raw array intensities—not just the genotypes—are made available. This is important because of the growing number of published algorithms that can convert raw SNP array data to copy number inferences.

— Tom LaFramboise

It must depend on the goals and types of each microarray study. While there are many other types and goals of microarray study, I illustrate three common cases: (1) discovery of differentially expressed genes between two or more tightly controlled experimental conditions. In this kind of study, if high-quality commercial microarrays are used, the number of replicates, or sample size of experiment, can be very small—often less than five, and sometimes even two or three, replicates per condition. Note that the research should utilize careful statistical analysis methods designed for small-sample microarray data analysis in this case. (2) Discovery of differentially expressed genes between two subject or patient groups with different biological or disease phenotypes or outcome. A much bigger sample size is required in this kind of study in order to obtain appropriate statistical power, especially avoiding the multiple comparisons pitfall due to a large number of false positives. A much bigger number, at least more than 15 different biological subjects or patients, would be needed for each group. (3) Discovery of gene prediction or classification models for close disease groups. One may need quite a large sample size of patients (and microarrays) to construct two completely independent subsets for model training (at least 30) and test (at least 20) sets.

— Jae Lee

Sample size is crucial because it determines statistical power. The weaker the effect one expects to find, the larger the sample size required. As most complex traits are pieced together from small effects, thousands of subjects will be typically required. The size of a genetic effect depends on the relative risk conferred and on the minor allele frequency. Genome-wide LD scanning mostly aims to detect weak-to-modest effects by reasonably common alleles. The "common disease-common variant" hypothesis postulates that most or all of genetic risk for common diseases is conferred by alleles common in the general population.

Toward a rough first estimate of genetic effect sizes to expect, a researcher must examine the heritability of the trait in question. Phenotypic homogeneity is crucial in estimating as well as optimizing effect sizes. Diseases often come with different forms, grades of severity, ages of onset, etc., and it is not safe to assume that the genetic underpinnings are the same. Finally, existing results from linkage studies may be useful in placing an upper bound on the expected effect sizes. Once a reasonable guesstimate has been made of the expected RR range, one can easily predict the sample size required to detect any combination of RR and MAF, at a given significance level.

— Constantin Polychronakos

With information about variation from one subset of each sample to another, we can actually calculate power. With experience, we have been able to use variation from prior assays to predict variation in upcoming assays with relative reliability, and thus to use standard power calculations.

— George Uhl

We always do each sample/treatment in duplicate to assure that the effects seen are real. These replicates are never technical replicates (same extraction divided in aliquots, etc.) but biological ones (two dishes of same cells treated separately).

We try to have enough biological variability by having at least three different cell lines/specimens to analyze, in order to capture some of the differences due to individual more than disease.

To assure that the effects we see are disease specific, we always use patient-matched germ line DNA (blood) to compare to the tumor. This allows us to screen out patient-specific changes.

— Jean Claude Zenklusen

Q3: What are your requirements in selecting an appropriate control group?

In an association study, the control cohort must be from the same population as the case cohort. Otherwise, population substructure may cause many false positive associations. Even if the case and control cohorts appear to be from the same population, it is a good idea to check for population substructure. There are several methods for identifying population substructure in the samples. These include applying tools such as STRUCTURE and EIGNESTRAT.

— Eleazar Eskin

Here, all of the usual epidemiological rules apply, e.g. the controls should be matched to the cases to the greatest degree possible. Having said that, though, there are currently efforts underway to make array data from "controls" publicly available. Recently, a group at the NCI published a case-control study that identified prostate cancer risk variants on chromosome 8q using the Illumina platform. They have plans to post all control (and case) genotype data on their Cancer Genetic Markers of Susceptibility web site. Efforts such as this to make banks of controls openly accessible to the community will greatly facilitate association studies.

— Tom LaFramboise

It must also be based on the scientific question of interest. One may be interested in the comparison between healthy versus tumor samples, between superficial versus invasive tumors, between atherogenic-prone mice under Chow versus Western diets, or between wild-type versus mutant cell lines. Some of the requirements in selecting control groups would be: (1) any confounding factors that affect the study results but are irrelevant (or uninteresting) to the main factor of investigation (e.g. when interested in the difference between smokers versus nonsmokers, any bias in gender between the two groups); (2) similarity of biological conditions and sample types (e.g. LDL-challenged macrophage versus oxLDL-challenged macrophage cells, rather than monocyte versus macrophage cells); and (3) availability of balanced sample sizes.

— Jae Lee

Matching cases and controls for every characteristic other than presence or absence of the disease is crucial in order to avoid population stratification. Geographic proximity has been established as the most important predictor of allele frequency in loci that differ among populations. The largest differences are found among continents but gradients exist even within the borders of a single country. However, to minimize the sacrifice in power such corrections entail, self-declared ancestry should be used to exclude individuals from different continents. A more subtle and difficult-to-correct source of stratification is the intra-continental allele gradients created by environment-driven strong and recent positive selection. Such loci are relatively few but very likely to show up as false positives when the entire genome is interrogated.

Balancing cases and controls for sex is important. Matching for age, sex, and environmental factors known to affect the illness is important in common diseases to avoid including among the controls many subjects who will later develop the disease (or would have, if exposed). It is less important with low-prevalence diseases.

Identification of controls should, ideally, be based on general-population databases, if individuals visiting the recruiting health facility are a random sample, not always a safe assumption. Health facility-based controls may be a good choice as long as a large enough variety of diagnoses is included and no single diagnosis makes up a substantial part of the cohort.

Finally, the use of parents has the advantage of minimizing or entirely avoiding population stratification through the use of family-based association analysis that uses the parents' untransmitted alleles as controls or the transmission disequilibrium test, examining divergence from the expected 50 percent of allele transmission from heterozygous parents.

— Constantin Polychronakos

Control groups that represent extreme phenotypes (super-normals) may be appropriate to enhance signals in initial gene-finding phases. Subsequently, it is useful to have population based controls (and disease samples that are representative of the disease in the population) in order to provide good estimates of population attributable risk.

— George Uhl

We have two control groups that are always included:

• Patient-matched germ line DNA to screen out intrinsic copy number/allelic variations.

• "Normal" samples, to separate organ-specific characteristics, not related to disease. These are more difficult to come by since normal brain tissue is not readily available (for obvious reasons), thus we have a lower number of cases for these (in our case epileptic frontal lobe resections, not normal, but at least non-tumoral).

Both types of controls have to be present in order to draw any meaningful conclusions.

— Jean Claude Zenklusen

Q4: How do you correct p-values for multiple hypothesis testing?

The simplest method is the Bonferroni correction. To apply this method, simply multiply the SNP's p-value by the number of SNPs that were genotyped. For example, if the best p-value is 10-8 and you collected 1,000 SNPs, then the Bonferroni adjusted p-value is 10-5. The Bonferroni method assumes that the SNPs are not correlated, which is not true. This results in adjusted p-values which are conservative, meaning that the p-values will appear as less significant than they actually are. A more accurate, but more computationally intensive, method for correcting for multiple hypothesis testing is using the permutation test. An implementation of the permutation test is available in the PLINK software.

— Eleazar Eskin

Given the complicated correlation structure among SNP markers, multiple hypothesis correction is difficult to do without being overly conservative. The vast majority of studies seem to perform a simple Bonferroni correction, which is typically far too conservative. Alternatively, our group often uses permutation testing to assign appropriate p-values to the observed test statistics. However, these permutation tests must be performed with care, as the quantities permuted must be exchangeable under the null hypothesis. When performed incorrectly, permutation testing can falsely inflate p-values. When in doubt, it is best to consult a statistician.

— Tom LaFramboise

In the large screening microarray studies, such a p-value correction should be based on the false discovery rate, which simultaneously controls false positive and false negative error rates and provides a practical cutoff criterion for further biological investigations and experiments. More recent resampling-based FDR estimation methods such as q-value and others would provide much more accurate and less conservative FDR values for correcting p-values for multiple comparisons.

— Jae Lee

Rather than correct the p-value itself, it is better to adjust the threshold at which the value is declared significant. If, for any reason, the value observed must be adjusted for n independent observations, the correct formula is:

Padj=1-(1-Pobs)n.

— Constantin Polychronakos

Monte Carlo simulations use the actual datasets generated, so that they do not require assumptions about underlying distributions of the data. They provide a technical control for us, since the same Perl scripts that are used to analyze the actual data can be used for the simulated datasets. Actual empirical p-values are generated. The disadvantage is that these simulations take relatively large amounts of time to run on relatively large workstations. False discovery rate corrections provide another approach; we should note that the different approaches to FDR correction that are currently available can yield differing p-values.

— George Uhl

We use a false discovery rate correction with a 0.05 threshold.

— Jean Claude Zenklusen

Q5: How do you establish thresholds to declare statistically significant association?

Establishing a significance threshold applies the same techniques as correcting for multiple hypothesis testing. First, we choose an overall significance threshold that we want to achieve, for example, a false positive rate of 0.05. If we are collecting 1,000 SNPs, then the Bonferroni significance threshold will be 0.00005. We can also obtain a significance threshold using a permutation test which will provide more accurate thresholds at a higher computational cost using software such as PLINK.

— Eleazar Eskin

The 0.05 threshold is the pervasive convention, but this is arbitrary. A more sensible approach seems to be to quote the uncorrected p-value when its corrected value is fairly low. Another approach that has gained favor in recent years is the q-value, which controls the false discovery rate rather than the false positive rate, and is generally makes more pragmatic sense in genome-wide scans than the p-value. A conventional threshold for the q-value is 0.25. This can be interpreted to mean that, if one performs a study using this threshold, 25 percent of the candidate regions identified as significant will be false leads.

— Tom LaFramboise

It would generally be acceptable if the threshold of a discovery method meets FDR < 0.1 (sometimes even < 0.2). If the number of identified targets is too big to further investigate with the above cutoff criterion, it can be further tightened to obtain a manageable number of statistically significant targets.

— Jae Lee

A p-value must satisfy the adjusted threshold: αβ=1-(1-α)1/n, where αβ is the Bonferroni-corrected alpha level that the p-value must satisfy to be considered significant at that level (typically α=0.05) after n observations. For the very high values of n and very low values of αβ seen in GWA designs, an excellent approximation is αβ=α/n. Dividing 0.05 by 500,000 gives 10-7. To obtain such low p-values with weak effects, large sample sizes are required.

However, this strict Bonferroni correction is too conservative. Because of LD, the n hypotheses are not independent (i.e. fewer than n hypotheses are effectively being tested). This is a problem even with arrays that minimize redundancy by using only tag SNPs, and more so with random-SNP arrays. Permutation analysis gives a better idea of what the exact significance threshold should be. However, the difference this makes is relatively small. Suppose that you are effectively testing n/3 instead of n hypotheses: the threshold is still a demanding 3 x 10-7. To reach such low p-levels with permutation analysis, the genotypes must be permuted ten million times for each of half a million markers a computationally demanding proposition.

Somewhat less obvious multiplicity of hypotheses must also be corrected for. A more subtle case of multiple-hypothesis testing is when genotype frequencies are "corrected" for covariates. Reporting the "best" result after trying a number of different combinations of covariates also multiplies hypotheses and requires correction.

In a two-stage design, the threshold must be calculated separately for each stage. Alternatively, a combined analysis of the two stages will require correction for the original number of hypotheses, to 10-7. Despite the lower threshold, combined analysis is more powerful under most circumstances.

— Constantin Polychronakos

We use the SNP chips as high-throughput genome surveying tools for copy number alterations and allelic imbalance studies (LOH, etc.). Still, we have to threshold to a minimal value to which we will consider an alteration, otherwise the whole genome shows up altered. For both types of analysis we use a 10 percent threshold, meaning that we will accept only changes that show in more than 10 percent of the samples being assayed. This may screen some minor players, but allows us to be certain that false positives are screened out.

— Jean Claude Zenklusen

Q6: What methods do you use to distinguish between false positives and true positives or noise?

We also make use of biological evidence, previously published studies, etc. For example, we are more likely to follow up a region with marginal statistical significance but previous annotation from the literature than we are a region with stronger statistical significance but with no associated publications.

— Tom LaFramboise

The first filter would be the FDR cutoff criterion. The candidate targets that meet such a statistical cutoff criterion should then be evaluated with their biological relevance. This can be started simply with their annotation and functional information search, and then based on certain pathway databases and tools such as KEGG, Ingenuity Pathway Analysis and GenMAPP. The targets identified with the most meaningful and relevant biological information would be confirmed by RT-PCR or other experimental techniques.

— Jae Lee

In addition to random noise, false positives in a GWA can result from a variety of biases. The most obvious is population stratification. Fortunately, half a million genotypes tell much about a subject's ancestry. Subjects with genetic material from outside the recruitment continent are best removed from further analysis using the STRUCTURE algorithm. This, however, offers no protection against MAF gradients within continents. A first step would be to test positive loci for evidence of recent positive selection, using the Haplotter tool. Evidence of such does not necessarily invalidate the locus (in fact, positively selected loci are more likely to have important allelic functions and are prime candidates for complex traits) and neither does absence of it rule out stratification. A more refined way of detecting and correcting stratification can be based on principal component analysis, one implementation of which for GWA is Eigenstrat. This algorithm models ancestry differences between cases and controls along continuous axes of variation and makes it possible to apply a correction specific to the MAF variation at each locus across ancestral populations, after removing obvious outliers in a manner that introduces no bias.

Markers with differences in call rates between cases and controls, makers with low overall call rates (e.g. less than 95 percent) or markers off HWE, should be excluded from further analysis. Finally, despite taking all of the above precautions, a careful look at the distribution of the χ2 statistic for all markers is crucial and will often reveal a divergence from that expected under the null hypothesis. Under the reasonable assumption that true susceptibility loci constitute only a tiny fraction of the total, such divergence can be assumed to represent unrecognized and uncorrected bias. Shifting the distribution by the empirical factor required to bring it back to "expected" is a crude but effective way of eliminating most bias with relatively little loss of power.

— Constantin Polychronakos

We have again been impressed by the likelihood that few single samples will be able to provide confidence in separating true- from false-positives. Independent observations made carefully in multiple samples provide the basis for confidence in individually identified results, just as they do for groups of results. We have routinely used three other controls to provide additional evidence for the chance that specific results may represent "noise." Large datasets from several groups of "control" individuals provide the opportunity to select SNPs that distinguish racial/ethnic groups. We can thus apply tests for the convergence between observed results and these SNPs as a test to see if racial/ethnic stratification "noise" in samples contributes to observed results, again using Monte Carlo simulations.

We have also kept track of the SNPs that provide the largest amount of technical noise in assays, again testing whether observed results overlap with these sets of technically noisy SNPs to greater extents than chance.

Finally, in assessments made using arrays of several different types, we have imposed requirements that results be supported from SNPs that lie on at least two different array types, to try to separate noise due to anomalies in just one hybridization probe synthesis.

— George Uhl

List of Resources

To solve even more microarray analysis problems, you may want to take a peek at the following resources. Our experts recommended several journal articles and online tools that may help you get the statistical power and significance that your research demands.

Publications

Barrett JC, Cardon LR. (2005). Evaluating coverage of genome-wide association studies. Nat Genet. 38(6):659-62

Itsik P, de Bakker PIW, Maller J, Yelensky R, Altshuler D, Daly MJ. (2006). Evaluating and improving power in whole-genome association studies using fixed marker sets. Nat Genet. 38:663.

Jain N, Thatte J, Braciale T, Ley K, O'Connell M, Lee JK. (2003). Local-pooled-error test for identifying differentially expressed genes with a small number of replicated microarrays. Bioinformatics. 19(15):1945-51.

Lange C, DeMeo D, Silverman E, Weiss S, Laird NM. (2004). PBAT: tools for family-based association studies. Am J Hum Genet. 74:367-9.

Pastinen T, Hudson TJ. (2004). Cis-acting regulatory variation in the human genome. Science. 306(5696):647-50.

Peng B, Kimmel M. (2007). Simulations provide support for the common disease-common variant hypothesis. Genetics. 175(2):763-76.

Pritchard JK. (2001). Are rare variants responsible for susceptibility to complex diseases? Am J Hum Genet. 69:124-3

Purcell S, Cherny SS, Sham PC. (2003). Genetic Power Calculator: design of linkage and association genetic mapping studies of complex traits. Bioinformatics. 19(1):149-150.

Redon R et al. (2006). Global variation in copy number in the human genome. Nature. 444(7118):444-54.

Skol AD, Scott LJ, Abecasis GR, Boehnke M. (2006). Joint analysis is more efficient than replication-based analysis for two-stage genome-wide association studies. Nat Genet. 38(2):209-13

Slager SL, Schaid DJ. (2001). Evaluation of candidate genes in case-control studies: a statistical method to account for related subjects. Am J Hum Genet. 68:1457-62.

Storey JD, Tibshirani R. (2003). Statistical significance for genome-wide studies. PNAS. 100(16):9440-5.

Websites

EIGNESTRAT
http://genepath.med.harvard.edu/~reich/EIGENSTRAT.htm

GenMAPP: Gene Map Annotator and Pathway Profiler
http://www.genmapp.org/

Genetic Power Calculator
http://pngu.mgh.harvard.edu/~purcell/gpc/

Haplotter
http://hg-wen.uchicago.edu/selection/haplotter.htm

PBAT: Tools for the statistical analysis of family-based association studies
http://www.biostat.harvard.edu/~clange/default.htm

PLINK: Whole genome association analysis toolset
http://pngu.mgh.harvard.edu/~purcell/plink/

Tagger: Selection and evaluation of tag SNPs
http://www.broad.mit.edu/mpg/tagger/

Structure
http://pritch.bsd.uchicago.edu/structure.html