# Quality Control of Genotypes Using Heritability Estimates of Gene Content at the Marker

- Natalia S. Forneris*,
^{††}, - Andres Legarra
^{†},^{‡},^{1}, - Zulma G. Vitezica
^{†},^{‡}, - Shogo Tsuruta
^{§}, - Ignacio Aguilar**,
- Ignacy Misztal
^{§}and - Rodolfo J. C. Cantet*,
^{††}

^{*}Departamento de Producción Animal, Facultad de Agronomía, Universidad de Buenos Aires, C1417DSE Buenos Aires, Argentina^{†}INRA, Génétique, Physiologie et Systèmes d’Elevage (GenPhySE), F-31326 Castanet-Tolosan, France^{‡}Université de Toulouse, INP, ENSAT, Génétique, Physiologie et Systèmes d’Elevage (GenPhySE), F-31326 Castanet-Tolosan, France^{§}Animal and Dairy Science, University of Georgia, Athens, Georgia 30602^{**}Instituto Nacional de Investigación Agropecuaria, Canelones 90200, Uruguay^{††}Consejo Nacional de Investigaciones Científicas y Técnicas, Av. Rivadavia 1917, C1033AAJ Buenos Aires, Argentina

- 1Corresponding author: INRA, UMR GenPhySE, CS52627, F-31326 Castanet-Tolosan, France. E-mail: andres.legarra{at}toulouse.inra.fr

## Abstract

Quality control filtering of single-nucleotide polymorphisms (SNPs) is a key step when analyzing genomic data. Here we present a practical method to identify low-quality SNPs, meaning markers whose genotypes are wrongly assigned for a large proportion of individuals, by estimating the heritability of gene content at each marker, where gene content is the number of copies of a particular reference allele in a genotype of an animal (0, 1, or 2). If there is no mutation at the marker, gene content has an additive heritability of 1 by construction. The method uses restricted maximum likelihood (REML) to estimate heritability of gene content at each SNP and also builds a likelihood-ratio test statistic to test for zero error variance in genotyping. As a by-product, estimates of the allele frequencies of markers at the base population are obtained. Using simulated data with 10% permutation error (4% actual error) in genotyping, the method had a specificity of 0.96 (4% of correct markers are rejected) and a sensitivity of 0.99 (1% of wrong markers are accepted) if markers with heritability lower than 0.975 are discarded. Checking of Mendelian errors resulted in a lower sensitivity (0.84) for the same simulation. The proposed method is further illustrated with a real data set with genotypes from 3534 animals genotyped for 50,433 markers from the Illumina PorcineSNP60 chip and a pedigree of 6473 individuals; those markers underwent very little quality control. A total of 4099 markers with *P*-values lower than 0.01 were discarded based on our method, with associated estimates of heritability as low as 0.12. Contrary to other techniques, our method uses all information in the population simultaneously, can be used in any population with markers and pedigree recordings, and is simple to implement using standard software for REML estimation. Scripts for its use are provided.

- gene content
- quality control
- SNP
- genomic selection
- REML
- shared data resource
- GenPred

IN PLANT and animal genetics, a large number of platforms for genotyping of single-nucleotide polymorphisms (SNPs) have appeared in recent years, in addition to the use of techniques to impute from low-density to high-density chips. However, these techniques are not without technical failures. Errors in genotypes can be due to wet laboratory errors (poor DNA samples, poor readings, etc.), different biochemistry in marker panels, label switching, or mistakes in pedigree recording. For example, Wiggans *et al.* (2012) removed 127 markers out of 2886 in the Bovine3K BeadChip (Illumina, Inc., San Diego, CA) because they showed >2% Mendelian conflicts. Errors also can arise from imputation procedures; for instance, if a marker is erroneously located in the map, its flanking markers will be wrong, as will be the imputation analysis (Hickey *et al.* 2012; Wang *et al.* 2013). The quality of genotypic data in genomic evaluations thus has been carefully considered for some time, and a number of procedures for quality control (QC) have been developed. The QC filtering of SNPs in genomic evaluation can increase accuracy, reduce computational effort, and improve stability of estimates of the effects of the remaining SNPs (Wiggans *et al.* 2009). We propose a method based on maximum likelihood to check the quality of marker genotypings in a possibly complex pedigreed population that is partially or completely genotyped for a set of biallelic markers. This method specifically aims at detecting loci for which a large number of individuals are wrongly genotyped. First, we briefly describe current methods. Then the method is presented, and results using publicly available and simulated data are shown.

## Quality Control of Genotypes

The most commonly used QC filters include QC on individuals for call rate, duplicates, and parent-progeny conflicts and QC on SNPs for call rate, minor allele frequency (MAF), departure from Hardy-Weinberg equilibrium, and in particular, Mendelian conflicts [see Wiggans *et al.* (2009) for a general description]. The latter are usually checked using data from trios and parent-offspring pairs (Wiggans *et al.* 2009, 2012). However, Mendelian-consistent errors usually go undetected (*e.g.*, an offspring *Aa* from parents *Aa* and *AA* is genotyped as *AA*). Another method involves checking that the progeny of one heterozygote male mated to several females has an average heterozygosity of 0.5 (Leroy *et al.* 2013).

Cheung *et al.* (2014) proposed a method for detecting both Mendelian-inconsistent and occasionally Mendelian-consistent errors tailored to small and moderately large human pedigrees (*e.g.*, 100 subjects). While this and similar error-detection procedures are computationally efficient for detecting genotyping errors at the marker level given inferred pedigree descent patterns and may allow tracing these occasionally occurring genotyping errors in markers at the subject level, it is not immediately known whether this method is equally applicable to plant or livestock data that are very large in pedigree size (*e.g.*, >1000) and especially when the motivation is to detect suspicious markers in which a considerable proportion of subjects has genotyping errors.

The application of these QC filters (except Cheung *et al.* 2014) does not use all available information from pedigree and markers. For instance, consider 10 full sibs, 5 with genotype *AA* and 5 with genotype *aa*, issued from both parents heterozygotes. This segregation distortion is not a Mendelian error, but it is a very unlikely situation. The problem becomes very complex for large pedigrees in which only a fraction of the animals is genotyped. For instance, VanRaden (2008) considered a pedigree with 3000 genotyped bulls, all connected, whose pedigree spanned 23,105 individuals.

Here we present a practical method to identify low-quality SNPs across individuals by considering gene content as a quantitative trait and testing the null hypothesis *h*^{2} = 1. The sensitivity and specificity of the method are evaluated by simulation of a pig breeding data set, and the method is illustrated with a real pig breeding data set.

## Materials and Methods

### Theory of the Method

#### Gene content as a quantitative trait:

Gene content *z* at one marker is the number of copies of a particular reference allele (*e.g.*, *z* = 0, 1, or 2 for *AA*, *AG*, and *GG*) (Falconer and Mackay 1996). In other words, (observed) gene content can be seen as a quantitative trait where the map of genotype to phenotype is for the three genotypes and the additive effect *a* of the reference allele (*G* in the preceding example) is exactly 1. Therefore, there is neither dominance nor epistasis. In addition, unless there is a mutation at the marker, and if the marker is genotyped accurately, there is no error associated with the phenotype. Thus, and by construction, the heritability of gene content is 1, and all variation is strictly additive genetic. The mean of *z* in the base population is 2*p*, where *p* is the allelic frequency at the base population, whereas its variance is 2*pq* and *q* = 1 − *p*.

The covariance between both gene contents from two individuals is [Cockerham 1969, equation (8)], where is the additive relationship between two individuals, usually computed from pedigree [see Toro *et al.* (2011) for a more detailed explanation]. This fact has been highlighted recently and used by McPeek *et al.* (2004) and Gengler *et al.* (2007) in similar contexts. Therefore, if **z** contains gene content for a set of genotyped individuals, , where is a matrix with additive relationships across genotyped individuals. Moreover, is a submatrix of the whole-pedigree relationship matrix **A**. For each marker, we may write a linear model for gene content: , where **u** is the deviation of each individual from this mean, and **e** is an error term that should be 0 in the absence of genotyping errors. In this case, so that is equal to 1 with no genotyping errors. We recall that and .

#### Estimation of heritability:

When analyzing complex pedigrees, a common method to estimate heritabilities is restricted maximum likelihood (REML) (Patterson and Thompson 1971). REML estimators were developed assuming normality, a situation that does not arise for gene content, which is not a continuous trait. However, REML has optimal properties as an iterated minimum-variance quadratic unbiased estimator (known as MIVQUE), which has minimum variance (Searle *et al.* 1992). The assumption of multivariate normality for gene content is a rather common one (McPeek *et al.* 2004; Gengler *et al.* 2007) and leads to a very convenient linearization of the problem. In particular, REML algorithms have two nice features for our purposes. The first is that they use Henderson’s mixed-model equations, which in this case are of the formwhere **W** is a matrix that contains **1** if the individual has a genotype and **0** otherwise, **z** contains observed genotypes (0, 1, 2) in a similar manner, **u** is expanded to include all individuals in the pedigree (Gengler *et al.* 2007), and . This corresponds to a linear model . This formulation, including animals with no genotype, allows use of the whole-pedigree matrix (Henderson 1977), which is very sparse and can be easily obtained using Henderson’s (1976) rules, which are computationally convenient with respect to the equations of McPeek *et al.* (2004) . From the final output, an estimate of *p* is obtained as . Another estimate that is slightly different because of numerical maximization of *p* (and *q =* 1 − *p*) is obtained as the solutions to the equation , *i.e.*, .

#### Hypothesis testing of genotyping errors:

Another feature of REML is that it computes likelihoods, from which statistic tests can be constructed. In our case, there are two hypothesis: the null hypothesis states that there are no genotyping errors, and therefore, (or ). The alternative hypothesis allows any positive value of the error variance. A likelihood-ratio test (LRT) can be used to reject the null hypothesis as follows: under the null hypothesis of zero genotyping error variance, the LRT statistic is asymptotically distributed as 1/2χ^{2}(0) + 1/2χ^{2}(1) (Self and Liang 1987; Visscher 2006). *P*-values for the observed LRT statistic can be calculated assuming this distribution. Although this assumes normality, LRT is known to be robust to departures from normality (*e.g.*, Almasy and Blangero 1998).

#### Implementation:

In practice, the method is simple. For each marker, REML estimates of and are obtained together with the value of the maximum likelihood—this is the alternative hypothesis. Another estimate is obtained with (in practice, set to a very small value). Later, the *P*-value of the LRT is computed from the two likelihoods, and a rejection threshold is established based on the preceding asymptotic distribution and a desired Type I error (in this work, 1%). We also suggest, as a less formal procedure, an inspection of the estimated heritability; heritabilities lower than 1 are suspicious.

#### Test in absence of pedigree:

If a pedigree is not available but most markers are *a priori* correctly genotyped, we suggest the following untested procedure:

After QC based on Hardy-Weinberg equilibrium, call rates, and MAF, construct a genomic relationship matrix

**G**using all markers,*e.g.*, following VanRaden (2008).Test the heritability of each marker using REML estimators as earlier and matrix

**G**(this procedure is sometimes called GREML) for the covariance of gene content across individuals.Discard markers rejected by the test, and iterate the procedure until no more markers are removed.

This procedure assumes that most markers are correct.

### Tests of the Method

#### Simulations:

To ascertain the sensitivity (*i.e.*, fraction of incorrect markers that are rejected) and the specificity (*i.e.*, fraction of correct markers that are *not* rejected) of the test, we simulated data using QmSim (Sargolzaei and Schenkel 2009) following Mendelian rules—therefore, the simulated data had no errors. We considered a simplified pig nucleus breeding program scenario with 10 autosomal chromosomes of 160 cM each and 70,000 SNPs. First, a mutation-drift equilibrium was reached in 2500 generations of random mating in a population with effective population size equal to 500 and mutation rate 2 × 10^{−4}, followed by a severe bottleneck with effective population size of 75 evolving during 30 generations. Then there was selection (not described here) during 5 generations, where 20 boars were mated with 200 sows producing 2000 offspring, with a total of 10,220 animals, with complete pedigree and 5000 randomly chosen animals genotyped for 28,254 markers with MAF > 0.01.

Type I error was evaluated as the number of SNPs with *P*-values above the significant level of 0.01 for the heritability test. We assessed type II error under two genotyping error scenarios by permuting both 10 and 5% of the genotypes for each SNP. Permuting the genotypes preserves minor allele frequencies and the Hardy-Weinberg equilibrium. Permuted genotypes were random for each SNP. Actually, a permuted genotype can be replaced by a correct genotype just by random, so the number of actual errors is lower than these values and a function of the allelic frequencies as follows: in Hardy-Weinberg equilibrium, a heterozygous genotype has a frequency of , and it is permuted by another heterozygous genotype with probability *x* (of being permuted) times (the frequency of another heterozygote). Extending the reasoning to the three possible genotypes, the rate of error is , a quartic in the allelic frequency. Thus, the actual error is 0.625*x* for a frequency of 0.5, 0.47*x* across a uniform spectrum of allelic frequencies, and lower for U-shaped distributions; in our particular simulation, the actual errors were 0.02 and 0.04 for the 5 and 10% permutation rates.

#### Data:

We used a pig data set that has been made available to the scientific community (Cleveland *et al.* 2012). The data set consisted of 3534 animals from a single PIC nucleus pig line with genotypes from the Illumina PorcineSNP60 chip (Ramos *et al.* 2009) with very little QC and a pedigree tracing back two generations from the genotyped animals (*N* = 6473). In practice, this data set should undergo Mendelian checking of parent-offspring couples to eliminate inconsistent animals; we have preferred not to do so in order to use the data set “as is.” A total of 50,433 SNPs were used in this study after filtering genotypes for minor allele frequency (<0.01) and the SNP call rate (<90%) and excluding SNPs on the sexual chromosomes. The reason to exclude MAF < 0.01 is that numerical maximization of REML is unreliable in that case. For comparison purposes, the same analysis was carried out in two extreme scenarios by randomly permuting half or all the genotypes for each SNP in the data set.

#### Statistical analysis:

For each SNP in the data set, the heritability and the LRT statistic were calculated to test for zero error variance in genotyping. The maximum residual log-likelihoods under the full (alternative hypothesis without *a priori* on the values of and ) and reduced models (null hypothesis assuming ) were obtained using remlf90 (Misztal *et al.* 2002). Heritability of gene content was estimated for each SNP under the full model. Estimates can be carried on in parallel if needed. Because this software uses Henderson’s mixed-model equations, the reduced model had to be approximated by specifying a very small value for the residual variance, . The advantage of computing in this way is that standard REML software can be used. We also used QC checking based on Mendelian errors included in preGSf90 (Aguilar *et al.* 2014); a marker was rejected if its genotypes showed Mendelian inconsistencies in more than 1% of the parent-offspring couples or trios.

The complete scripts for QC, the PIC data set, and the three simulated data sets are available at http://genoweb.toulouse.inra.fr/~alegarra/qualitycontrol.tar.gz. A version with the scripts and a small subset of the PIC data set is available as Supporting Information, File S1.

## Results

### Simulations

Figure 1B displays estimates of heritability (Figure 1A shows *P*-value of the LRT) as a function of minor allele frequency for the data simulated with no error. Most heritability estimates are very close to 1, even for very low MAF values, although there is a trend that markers with very low MAF values have lower heritabilities (*e.g.*, they may become fixed by drift). Using a threshold at nominal 0.01 type I error, the sensitivity when 10% (5%) of the simulated genotypes were erroneous (permuted at random) was 0.99 (0.91). The specificity was 0.95 (*i.e.*, 5% of correct SNPs were rejected). Alternatively, we bounded the estimates of heritability for rejection. Figure 1C shows type I (or 1-specificity) and type II (1-sensitivity) errors against putative thresholds for rejection based on heritability. For instance, if markers are rejected if their heritability estimate is lower than 0.975, this results in a specificity of 0.96 (4% of correct markers are rejected) and a sensitivity of 0.99 (for 10% of permuted data, 1% of all wrong markers are accepted). Choosing a lower bound such as 0.90 results in only 0.04% of markers being incorrectly rejected but as much as 6.5% of markers being incorrectly accepted. These figures change with the level of quality, and the situation with 5% permutation of genotypes gives higher type II error. Checking Mendelian errors with preGSf90 performed worse, with 0 type I error (as expected) and 0.54 (0.84) sensitivity in the scenario where 5% (10%) genotypes were permuted. A receiving operator curve detailing results for no error *vs.* 10% error is available in Figure S1.

### Real Data

Figure 2A shows box plots for the estimated heritability of the SNPs for the original data set, the half-permuted data set, and the completely permuted data set. The original data set had a mean heritability of 0.99, and 75% of the SNPs had heritabilities above this value, although some of the markers deviated highly from 1. When half the genotypes for each SNP were randomly permuted, heritability ranged from 0.02 to 0.84, the mean heritability was 0.25, and 75% of the estimates were below 0.27. For the fully permuted data set, all heritabilities were below 0.07. The boxes shift upward as the overall quality of the data set improves. When testing the null hypothesis of zero genotyping error at *α* = 0.01, the null hypothesis was rejected in 8% (*N* = 4099) of the SNPs of the original data set, whereas all the *P*-values were below 10^{−12} for the half-permuted data set and below 10^{−93} for the fully permuted data set. The latter exemplifies how a genotyping procedure that is largely wrong for a large percentage of the individuals (>50%) can be easily spotted using our method. The 4099 markers that did not pass the test in the original data set should be declared wrongly genotyped, and their genotypes should not be used in later analyses. Figure 2B illustrates the relationship between heritability estimates and *P*-values of the LRT when REML is used to estimate variance components of the original data set. It can be seen that rejected SNPs had the lower estimates of heritability, although the range of values was large (0.13 < *h*^{2} < 0.97 for SNPs with *P* < 0.01). For example, a marker with a (very) low MAF can result in high estimates of heritability, but the LRT may be inconclusive because there is very little information in the data. In general, however, using either a formal LRT or estimates of heritability will produce similar results. Using heritability estimates is less formal statistically, but it is easy to interpret for quantitative geneticists, and it can lead to an easy speed-up of the method, as discussed later. In this data set, preGSf90 checking of Mendelian errors detected only one wrong marker at 1% tolerance for Mendelian inconsistencies, 577 at 0.1% tolerance, and 2019 at zero tolerance. Among the 577 markers with >0.1% conflicts, 388 were in common with the 4099 rejected by the heritability test. The 577 markers with >0.1% Mendelian inconsistencies had lower heritability estimates (0.92 on average) than those that were not rejected (0.99).

## Discussion

Our original method was successful in identifying low-quality markers in a complex pedigree. The method avoids finding pedigree structures such as father-son pairs or trios. Although the analysis of each individual marker takes a few minutes, it can be parallelized because each marker is independent. Our method provides a statistical test, and therefore, its properties are known, whereas for other procedures, cutoff thresholds are largely arbitrary. As shown by our results, other tests, such as the parent-offspring pairs, have high specificity but not necessarily high sensitivity, *e.g.*, if not many parent-offspring pairs exist. In addition, our method takes into account segregation distortions. The method seems to be robust to the presence of a low allele frequency (say, >0.05). However, for very low allelic frequency, estimates of heritability and LRT tend to be unreliable (Figure 1, A and B), although we have not tested the method for values lower than 0.01. Our procedure cannot correct Mendelian errors for markers that are not rejected, and in this case, the use of parent-offspring comparisons is necessary.

The method assumes a single population in Hardy-Weinberg equilibrium. The latter hypothesis seems not very restrictive because the simulated data included selection. If the population has different origins, still, . However, the hypothesis of common means and variances will not hold. An approximate method consists of fitting different origins using an unknown parent groups model (Quaas 1988), *i.e.*, allowing for different allelic frequencies at different base populations. This assumes the same value of across all populations, which will be true if the frequencies are similar across populations but will be false if there is a large divergence.

While REML estimation of heritability for a single SNP is likely to be fast, the total number of computations for a large number of SNPs can take days. In expectation-maximization REML (EM-REML), the most expensive operations in one round of iteration include (1) setting the mixed-model equations, (2) calculating solutions, and (3) calculating traces. However, the mixed-model equations are the same for a value of heritability in all markers. Thus, dramatic costs savings can be realized when factorizations and traces are precomputed for several values of heritability. If the purpose of QC is to select SNPs with, say, *h*^{2} > 0.98, only three sets of such matrices would be required, *e.g.*, at *h*^{2} = 0.99, 0.98, and 0.97.

Our procedure cannot identify pedigree errors (*i.e.*, mislabeling of DNA samples). In this case, errors are across markers in one individual instead of being across individuals for one marker. Parent-offspring discordances can flag such an error if many markers do not follow Mendelian rules for a given parent-offspring pair. There are procedures to assign parents (Wiggans *et al.* 2009; Hayes 2011; VanRaden *et al.* 2013). However, a general procedure to identify and correct pedigree errors does not exist yet. A practical procedure is to compare genomic relationships (VanRaden 2008) and pedigree relationships and inspect the differences, which depend on the relationship itself and the genome architecture. A thorough description of such differences can be found in Wang *et al.* (2014).

A particular case is the use of genotypes from different chips or panels, possibly with different chemistry, *e.g.*, the 50K and 3K panels in cattle (Wiggans *et al.*, 2012). These authors found that some markers were correctly read using one panel but not the other. In our method, this would be observed because heritability estimated including genotypes from the faulty chip, either alone or combined with the other panel, would decrease. This also applies to samples genotyped in batches; *e.g.*, if there is a (large) batch of individual samples with poor DNA conditions, the addition of genotypes from the sample will decrease heritability estimates.

In our experience, this procedure is most useful when dealing with new complete data sets, in particular, from experimental studies. Regular genetic evaluations, as in dairy cattle, keep a better track of DNA samples, and because of the abundance of parent-offspring couples and trios, poor-quality markers are easily found (Wiggans *et al.* 2009, 2012).

## Conclusion

We have introduced a practical QC procedure to identify SNPs with low quality across many individuals. The proposed filter is in essence an estimate of heritability of gene content at the SNPs, where any deviation from 1 is suspicious, and the *P*-value is for testing the null hypothesis of “no error in genotyping.” This QC procedure can jointly consider all genotyped individuals and their pedigree and uses standard hypothesis-testing procedures. It should be used as a complement to standard QC procedures and possibly after them.

## Acknowledgments

The editor and reviewers are acknowledged for very useful comments. This work was made possible by a visit of NS Forneris to INRA, Toulouse, France, financed by the Saint-Exupéry Scholarship Program 2013–2014 (MinCyT Argentina–French Embassy). AL and ZGV acknowledge financing from projects X-Gen and GenSSeq of INRA metaprogram SelGen. NSF and RJCC were partially funded by grants UBACyT 861/2011 and PIP CONICET 833/2013. This project was partly supported by the Toulouse Midi-Pyrénées bioinformatics platform.

## Footnotes

Supporting information is available online at http://www.genetics.org/lookup/suppl/doi:10.1534/genetics.114.173559/-/DC1

*Communicating editor: S. Sen*

- Received September 26, 2014.
- Accepted December 18, 2014.

- Copyright © 2015 by the Genetics Society of America