BACKGROUND
approximately one-half of the human genome consists of the remnants of past transpositional bursts  <cit> .
line- <dig> non-ltr retrotransposons and the alu elements they mobilize continue to replicate in the human gene pool to this day  <cit> .
as a result of alu retroposition, our genomes are littered with more than one million small , non-allelic regions whose dna sequences are nearly identical to each other.
their recombinogenic impact is evident: these scattered homologies trigger non-allelic homologous recombination  events that lead to translocations, deletions, duplications, and other chromosomal abnormalities and copy number variations  <cit> .
these events have affected the long-term evolution of the human genome and of the alu insertions themselves  <cit> .
alu repeats have been implicated in differential methylation states of the genome, in the translation response to cellular stress, and in the regulation of transcription  <cit> .
however, the impact of alu insertions on the rates of allelic recombination events in the human germline remains largely unknown.
it has been suggested that polymorphic alu insertions may suppress recombination when found in the heterozygous state  <cit> , and fixed alu insertions may contain specific dna sequence features capable of recruiting recombination-enhancing or -suppressing factors.
meiotic recombination rates in humans vary widely across the genome  <cit> .
the search for the causes of this variation initially focused on broad-scale dna sequence and chromosome-level features, such as g+c and cpg content, or the density of poly/poly stretches and protein-coding genes  <cit> .
although these features explain nearly half of the variance in recombination rate at the  <dig> mb scale, they explain less than 5% of the variance of recombination at the  <dig> kb scale  <cit> .
more recently, attention has turned to dna sequence motifs associated with recombination "hot spots," where many recombination events are concentrated  <cit> .
a family of short  hot spot-associated motifs may account for a sizable proportion of those hot spots and thus for a substantial proportion of the variance in recombination rate.
these motifs are common outside of alu elements and in other repeat sequences , but some alu elements carry those motifs  <cit> .
that association translates into a slight enrichment of several alu subfamilies in hot spots , and consequently an association with higher recombination rates  <cit> .
however, that effect appears to be due entirely to the recombinogenic motifs: to the extent tested, no association was found between alu insertions lacking the motifs and higher recombination rates  <cit> .
these negative results imply that the alu sequence is not uniquely nor highly recombinogenic in itself.
since previous studies have analyzed recombination rate variation at a broad scale, or have focused mainly on hot spots, a less dramatic effect , or an effect mediated only by a minority of more recently-inserted copies, would have gone undetected.
yet even if the impact of individual alu insertions on local recombination rates is small, the sum of those effects over the very large number of alu insertions in the human gene pool could have a significant cumulative impact on the structure of our genomes.
moreover, any effect of alu insertions on recombination rate in their immediate vicinity could influence their own evolutionary fates, the evolution of the alu retroposon family, and the evolutionary responses of the genetic pathways that regulate recombination itself.
here we focus specifically on the effect of recent  aluy insertions.
of all the repeat families in the human genome, the aluy subfamily has the largest number of recently inserted copies.
any alu-specific properties that affect recombination should be most apparent in young insertions, rather than older insertions that have accumulated many mutations that may have altered their properties.
the high copy number of aluy insertions provides the statistical power needed to detect modest effects, and the homogeneity of the subfamily reduces the danger of missing an effect due to heterogeneity within the data set.
our question is: does the presence of an aluy insertion affect the local rate of recombination?
we show that the presence of a fixed, young aluy insertion is significantly predictive of a modestly elevated local recombination rate.
RESULTS
in order to address the effect of alu insertions on local recombination rates as directly and clearly as possible, we sought to eliminate or account for factors and biases that could affect recombination rate estimates.
in short, we first constructed data sets that avoid complicating factors and biases and then used covariates in stepwise linear regression analyses to account for the remaining factors.
the basic unit in our analyses is a ~ <dig> kb region containing a single aluy insertion locus and common snps spaced at 4- <dig> kb intervals throughout each region.
the exact size of any particular "aluy region" is determined by the locations of the first and last snp ascertained for that region.
by focusing on regions with just one aluy insertion, we avoid modeling complex interactions between multiple aluy insertions in one or several inter-snp intervals.
by maintaining uniformity of inter-snp interval sizes, we avoid biases in the estimation of recombination rates on intervals of very different sizes.
the frequency of common snps in the human population and our need for uniformly-sized intervals across many aluy regions constrain our choice of snp spacing intervals.
under those constraints, the 4- <dig> kb snp spacing best meets our goal of estimating recombination rates in small intervals.
we used this same strategy to select aluy regions and uniformly-spaced snps from our own "world diversity panel"  and from the hapmap phase ii data.
after selecting aluy regions and snps within them, we used the genotypes at those snps in various population samples to estimate the rescaled recombination rate parameter  for each inter-snp interval.
a typical aluy region, with ρ estimates plotted for each inter-snp interval, is shown in figure  <dig>  the values of other covariates for each interval were computed as detailed in the methods section.
stepwise linear regression was used to ascertain whether the presence of an aluy insertion locus in an inter-snp interval significantly changes the recombination rate in that interval, relative to the rate in intervals that do not contain an aluy insert.
aluy regions in world diversity panel
we designed our first data by ascertaining evenly spaced common snps from a panel of samples drawn from africa, asia, and europe, then genotyping those snps in our population samples from those continental groups .
our stepwise linear regression analyses detected a significant positive effect  of the presence of a fixed aluy insertion on the local recombination rate in the african subset of our world population diversity sample .
as expected, both the regional mean recombination rate and the percent g+c in an interval significantly predicted the recombination rate.
the a priori expected effect of hot spots is slightly weaker and does not reach statistical significance.
no significant evidence of an effect of fixed aluy insertions on recombination was found in the east asian or european data subsets.
we also found no evidence that the five polymorphic aluy insertions influenced local recombination rates in african, east asian, or european population samples .
the means and standard deviations of the variables are shown in table  <dig>  terminal inter-snp intervals  were excluded from the regression analyses out of concern that their recombination rate estimates might be downwardly biased .
a number of individuals used to infer recombination rates, minimum to maximum .
b total number of inter-snp intervals analyzed for a data set.
a for each interval, the regional log <dig> is the weighted average taken over all intervals in the region, excluding that focal interval.
b aluy length statistics are given for descriptive purposes.
only the presence or absence of an aluy is used as a regression variable.
the statistical power of this data set of  <dig> fixed aluy regions and  <dig> polymorphic regions is limited to detecting large effects.
the significant association between aluy insertions and increased recombination observed in the african sample, but not in the non-african samples, likely reflects the earlier founding and larger effective population size of the african population  <cit> .
these attributes increase the number of detectable recombination events, and thus the statistical power to detect factors associated with recombination, in this population.
inter-snp interval length, recombination rate, and aluy insertions
to increase the power to detect any association between aluy elements and recombination rate, we used data from the hapmap project .
this large data set provides estimates of the inter-snp recombination rate for every inter-snp interval in the data  <cit> .
before making use of this resource, however, we examined the data set for biases that might impede our ability to detect an effect of alu elements on recombination.
our initial analyses of the hapmap data found that, in general:  longer-than-average inter-snp intervals have lower-than-average estimated recombination rates ;  inter-snp intervals with aluy insertions in them are longer than intervals without them; and  aluy insertions are associated with both longer-than-average intervals and lower-than-average estimated recombination rates.
specifically, among  <dig> , <dig> autosomal inter-snp intervals with lengths between  <dig> and  <dig>  bp for which recombination rates were estimated by the hapmap project, a linear regression of recombination rate  on interval length  yields a significantly negative slope .
of those intervals,  <dig>  contain at least part of an aluy insertion .
consistent with the overall pattern, those intervals are longer, on average, than intervals without an aluy insertion .
again consistent with the general pattern, the estimated recombination rate in these longer, aluy-containing intervals is lower than in the shorter intervals that lack aluy insertions .
the reason for the larger average size of intervals with an aluy in them  is unclear.
it might be due to the difficulty of designing robust genotyping assays for snps near repeat sequences in the context of a high-throughput genotyping project, or perhaps these repeats are in fact associated with lower nearby genetic variation.
we observed a similar pattern with line- <dig> insertions .
the cause of the general association between estimated recombination rate and inter-snp interval length in hapmap data may be an artifact of the estimation procedure, since regions of lower snp density contain less information about past recombination events.
we therefore eliminated the potentially confounding relationship between inter-snp interval size and estimated recombination rate by selecting uniformly sized intervals , re-estimating the recombination rates in those intervals, and also including interval length as a covariate in our regression analyses.
previous analyses of correlations between aluy insertions and local recombination rates did not account for this bias and were focused on larger effect sizes, which may explain why no aluy-specific effect was detected  <cit> .
aluy regions in hapmap yri trios
to further test the initial results we observed in the african sample of our world diversity panel, we assembled a genome-wide data set of hapmap snps typed on the  <dig> yoruba  parent-child trios  <cit> .
aluy insertions were identified by repeatmasker  and excluded known polymorphic insertions by comparison with dbrip  <cit> .
we selected  <dig>  aluy regions and uniformly-spaced snps within them  from the hapmap data using an adaptation of the method we used on our world diversity panel above .
we analyzed these data after removing the terminal and sub-terminal intervals from each region  to eliminate edge effects.
table  <dig> shows the means and standard deviations of log <dig> for these intervals and for the seven predictor variables defined for each inter-snp interval: length, regional recombination rate, g+c content, "core" motif count, "extended" motif count, hot spot presence, and aluy presence .
after accounting for all other effects in the model, we find that the presence of an aluy insertion predicts a statistically significant  though modest  increase in recombination rate.
the length of an interval has a significant but very small effect on the recombination rate in that interval, which implies that our strategy to eliminate the interval length factor was successful.
each of the other variables is independently predictive of the local recombination rate.
the impacts of the regional recombination rate and g+c composition are the largest and most significant, as expected.
the hot spot-associated recombinogenic motifs are associated with small, local variations in recombination rate independently of their association with hot spots.
since those motifs are present in a minority of hot spots  <cit> , it is not surprising that hot spots themselves have an independent and much stronger effect: an interval that overlaps a known hot spot has a  <dig> -fold greater recombination rate, on average, compared with nearby intervals that do not overlap a hot spot.
aluy regions in hapmap ceu trios
with our initial set of  <dig> aluy regions, we detected no effect of fixed aluy insertions on recombination in our european population sample.
we then asked the same question using  <dig>  aluy regions  and  <dig> ceu parent-child trios genotyped by the hapmap project.
means and standard deviations of the regression variables are shown in table  <dig>
regression analysis  shows that the presence of a fixed aluy insertion is associated with an ~8% increase in the recombination rate in an interval .
the overall results are very similar to those observed with the yri data.
this similarity is expected, since the patterns of variation and linkage disequilibrium in the ceu population sample are correlated with those in the yri sample because of their shared ancestry.
the effect of interval length on recombination rate is small, as it was with the yri data, and statistically insignificant in this case.
approximately 16%  of the aluy-containing intervals in the ceu data set also overlap a hot spot.
while that does not mean that those hot spots overlap the aluy insertions themselves, we nonetheless checked for a potential interaction effect between hot spots and aluy insertions, since such an interaction could account for part of the aluy effect.
we observed a significant interaction effect in the ceu hapmap data set .
independent effect sizes for aluy and hot spots were slightly but not significantly reduced .
both effects remain clearly significant, and the results for other factors are only trivially affected.
no significant hot spot × aluy effects were seen in the other data sets.
the differences between the results based on our world diversity panel and those obtained from the hapmap data should be considered with caution, due to the small number of regions genotyped in the former.
an effect size of ~8% would not be detectable with a sample size of  <dig> intervals in  <dig> aluy regions, so the negative results obtained in our european and east asian samples are probably due to a lack of statistical power.
although the 95% confidence intervals around the effect sizes estimated for the fixed aluy insertions in the sub-saharan african and the hapmap yri data sets do not overlap, the 99% confidence intervals do .
the majority of aluy insertions are 10- <dig> million years old .
the accumulation of mutations in aluy copies as they age might alter their effects on the local recombination rate.
figure  <dig> explores how the effects of aluy insertions on recombination depend on their degree of sequence divergence from their subfamily consensus sequences, and figure  <dig> shows the relative frequencies of aluy insertions plotted against their divergence.
a similar recombination rate effect is seen across all classes of the aluy divergence spectrum.
discussion
we have assessed whether aluy insertions affect the recombination rate in their immediate neighborhood.
we first generated and analyzed a data set of aluy insertions and surrounding snps that were ascertained to limit extraneous factors and thus to maximize our ability to detect such effects.
to test the observations gained from those data, we extended the ascertainment design and analyses to a larger set of aluy insertions and neighboring snps extracted from the hapmap phase ii data.
because aluy insertions are correlated with some sequence features  that are themselves associated with higher recombination rates or with recombination hot spots, we included those features as covariates in our analyses.
we included hot spots themselves as proxies for the as-yet-unknown factors that presumably cause those hot spots.
as expected, the average recombination rate within ~ <dig> kb on either side of an aluy-containing interval was a strong predictor of that interval's recombination rate.
while this yields no insight about the cause of that broad-scale variation, it allows us to factor out any effects at that scale.
even with the mean surrounding regional recombination rate already factored out, the g+c content of an inter-snp interval is strongly predictive of its recombination rate.
the g+c content itself is correlated with the "core" and "extended" hot spot-associated recombinogenic motifs, since they are gc-rich.
nonetheless, both of those motifs carry additional significant predictive power.
as expected, the presence of a hot spot in  an interval has a much stronger effect, increasing the recombination rate by ~ <dig> -fold, on average.
there is a slight association between hot spots and aluy insertions : inter-snp intervals that contain an aluy are 13% more likely to overlap a hot spot than control intervals are .
some degree of association would be expected under the hypothesis that aluy insertions increase the local recombination rate, since they would push that rate past the threshold for hot spots in at least some regions.
there is also some evidence for a positive interaction between hot spots and aluy insertions .
however, since many unknown factors may interact to generate recombination hot spots, and since an aluy-specific effect should be detectable independently from those factors and the hot spots they generate, we have attempted to factor out the effect of hot spots.
after factoring out effects that are not specific to aluy sequences, we still find that the presence of a fixed aluy insertion has a significant positive impact on the recombination rate within the ~ <dig> kb inter-snp interval that contains it.
a fixed aluy insertion appears to cause a twofold enhancement of the local recombination rate in the  <dig> aluy regions we genotyped in our sub-saharan african sample.
a smaller positive effect - a  <dig> % increase over the surrounding intervals, on average - is strongly evident in the larger hapmap-based data sets, for both the yri and ceu populations.
no relationship between polymorphic aluy insertions and the local recombination rate was found in the five regions genotyped in our world diversity panel, but a modest effect  would not be detectable in a data set of that size.
we therefore turned to the hapmap yri trio data set to test for a smaller effect of polymorphic aluy insertions on the local recombination rate.
using the methods we applied above to ascertain fixed aluy regions, we identified  <dig> polymorphic aluy regions based on the aluy loci in dbrip  <cit> .
we examined  <dig>  inter-snp intervals  and found no significant effect of the presence of polymorphic aluy elements on local recombination rates.
the magnitude of the per-copy effect of a fixed aluy on the local recombination rate is comparable to the effect of the stronger of the two recombinogenic motifs that we analyzed .
given the resolution of our data sets , it is possible that the effect may be stronger but more localized than we have reported, since the effect is diluted out over the entire aluy-containing interval.
in considering potential causes of the observed effect, it must be noted that the recombination rates estimated here reflect only the history captured by human snps, nearly all of which arose less than  <dig>  myr ago.
thus aluy characteristics that existed only prior to that time  cannot explain the recent effect of those insertions.
aluy sequences might bind cofactors or influence chromatin structure in a way that influences the local recombination rate, as has been suggested for some short recombinogenic motifs  <cit> .
for example, alu insertions are typically flanked on both sides by target sites for line- <dig> endonuclease.
this is because alu insertions are created by line-1-encoded proteins  <cit>  at line- <dig> endonuclease cutting sites, and the original target sites are duplicated during the insertion event.
alu insertions may thus attract line- <dig> endonuclease, which creates double-strand breaks  in the dna that can then be resolved as recombination events.
line- <dig> endonuclease generates large numbers of dsbs  <cit> , which suggests that endogenous line- <dig> activity might generate dsbs at a rate sufficient to affect recombination rates.
CONCLUSIONS
in summary, we have demonstrated that the presence of a fixed aluy element enhances the local recombination rate by approximately 6%.
this effect is similar in magnitude to that observed for previously identified recombinogenic motifs.
while the effect of each aluy element is relatively small, the presence of hundreds of thousands of these elements throughout the human genome implies that they exert a substantial effect on genome-wide recombination rates.
further research is needed to identify precisely the molecular mechanism responsible for this effect.

 approximately one-half of the human genome consists of the remnants of past transpositional bursts. line- non-ltr retrotransposons and the alu elements they mobilize continue to replicate in the human gene pool to this day. as a result of alu retroposition, our genomes are littered with more than one million small, non-allelic regions whose dna sequences are nearly identical to each other. their recombinogenic impact is evident: these scattered homologies trigger non-allelic homologous recombination events that lead to translocations, deletions, duplications, and other chromosomal abnormalities and copy number variations. these events have affected the long-term evolution of the human genome and of the alu insertions themselves. alu repeats have been implicated in differential methylation states of the genome, in the translation response to cellular stress, and in the regulation of transcription. however, the impact of alu insertions on the rates of allelic recombination events in the human germline remains largely unknown. it has been suggested that polymorphic alu insertions may suppress recombination when found in the heterozygous state, and fixed alu insertions may contain specific dna sequence features capable of recruiting recombination-enhancing or -suppressing factors. meiotic recombination rates in humans vary widely across the genome. the search for the causes of this variation initially focused on broad-scale dna sequence and chromosome-level features, such as g+c and cpg content, or the density of poly/poly stretches and protein-coding genes. although these features explain nearly half of the variance in recombination rate at the mb scale, they explain less than 5% of the variance of recombination at the kb scale. more recently, attention has turned to dna sequence motifs associated with recombination "hot spots," where many recombination events are concentrated. a family of short hot spot-associated motifs may account for a sizable proportion of those hot spots and thus for a substantial proportion of the variance in recombination rate. these motifs are common outside of alu elements and in other repeat sequences, but some alu elements carry those motifs. that association translates into a slight enrichment of several alu subfamilies in hot spots, and consequently an association with higher recombination rates. however, that effect appears to be due entirely to the recombinogenic motifs: to the extent tested, no association was found between alu insertions lacking the motifs and higher recombination rates. these negative results imply that the alu sequence is not uniquely nor highly recombinogenic in itself. since previous studies have analyzed recombination rate variation at a broad scale, or have focused mainly on hot spots, a less dramatic effect, or an effect mediated only by a minority of more recently-inserted copies, would have gone undetected. yet even if the impact of individual alu insertions on local recombination rates is small, the sum of those effects over the very large number of alu insertions in the human gene pool could have a significant cumulative impact on the structure of our genomes. moreover, any effect of alu insertions on recombination rate in their immediate vicinity could influence their own evolutionary fates, the evolution of the alu retroposon family, and the evolutionary responses of the genetic pathways that regulate recombination itself. here we focus specifically on the effect of recent aluy insertions. of all the repeat families in the human genome, the aluy subfamily has the largest number of recently inserted copies. any alu-specific properties that affect recombination should be most apparent in young insertions, rather than older insertions that have accumulated many mutations that may have altered their properties. the high copy number of aluy insertions provides the statistical power needed to detect modest effects, and the homogeneity of the subfamily reduces the danger of missing an effect due to heterogeneity within the data set. our question is: does the presence of an aluy insertion affect the local rate of recombination? we show that the presence of a fixed, young aluy insertion is significantly predictive of a modestly elevated local recombination rate.  in order to address the effect of alu insertions on local recombination rates as directly and clearly as possible, we sought to eliminate or account for factors and biases that could affect recombination rate estimates. in short, we first constructed data sets that avoid complicating factors and biases and then used covariates in stepwise linear regression analyses to account for the remaining factors. the basic unit in our analyses is a ~ kb region containing a single aluy insertion locus and common snps spaced at 4- kb intervals throughout each region. the exact size of any particular "aluy region" is determined by the locations of the first and last snp ascertained for that region. by focusing on regions with just one aluy insertion, we avoid modeling complex interactions between multiple aluy insertions in one or several inter-snp intervals. by maintaining uniformity of inter-snp interval sizes, we avoid biases in the estimation of recombination rates on intervals of very different sizes. the frequency of common snps in the human population and our need for uniformly-sized intervals across many aluy regions constrain our choice of snp spacing intervals. under those constraints, the 4- kb snp spacing best meets our goal of estimating recombination rates in small intervals. we used this same strategy to select aluy regions and uniformly-spaced snps from our own "world diversity panel" and from the hapmap phase ii data. after selecting aluy regions and snps within them, we used the genotypes at those snps in various population samples to estimate the rescaled recombination rate parameter for each inter-snp interval. a typical aluy region, with ρ estimates plotted for each inter-snp interval, is shown in figure the values of other covariates for each interval were computed as detailed in the methods section. stepwise linear regression was used to ascertain whether the presence of an aluy insertion locus in an inter-snp interval significantly changes the recombination rate in that interval, relative to the rate in intervals that do not contain an aluy insert. aluy regions in world diversity panel we designed our first data by ascertaining evenly spaced common snps from a panel of samples drawn from africa, asia, and europe, then genotyping those snps in our population samples from those continental groups. our stepwise linear regression analyses detected a significant positive effect of the presence of a fixed aluy insertion on the local recombination rate in the african subset of our world population diversity sample. as expected, both the regional mean recombination rate and the percent g+c in an interval significantly predicted the recombination rate. the a priori expected effect of hot spots is slightly weaker and does not reach statistical significance. no significant evidence of an effect of fixed aluy insertions on recombination was found in the east asian or european data subsets. we also found no evidence that the five polymorphic aluy insertions influenced local recombination rates in african, east asian, or european population samples. the means and standard deviations of the variables are shown in table terminal inter-snp intervals were excluded from the regression analyses out of concern that their recombination rate estimates might be downwardly biased. a number of individuals used to infer recombination rates, minimum to maximum. b total number of inter-snp intervals analyzed for a data set. a for each interval, the regional log is the weighted average taken over all intervals in the region, excluding that focal interval. b aluy length statistics are given for descriptive purposes. only the presence or absence of an aluy is used as a regression variable. the statistical power of this data set of fixed aluy regions and polymorphic regions is limited to detecting large effects. the significant association between aluy insertions and increased recombination observed in the african sample, but not in the non-african samples, likely reflects the earlier founding and larger effective population size of the african population. these attributes increase the number of detectable recombination events, and thus the statistical power to detect factors associated with recombination, in this population. inter-snp interval length, recombination rate, and aluy insertions to increase the power to detect any association between aluy elements and recombination rate, we used data from the hapmap project. this large data set provides estimates of the inter-snp recombination rate for every inter-snp interval in the data. before making use of this resource, however, we examined the data set for biases that might impede our ability to detect an effect of alu elements on recombination. our initial analyses of the hapmap data found that, in general: longer-than-average inter-snp intervals have lower-than-average estimated recombination rates; inter-snp intervals with aluy insertions in them are longer than intervals without them; and aluy insertions are associated with both longer-than-average intervals and lower-than-average estimated recombination rates. specifically, among, autosomal inter-snp intervals with lengths between and bp for which recombination rates were estimated by the hapmap project, a linear regression of recombination rate on interval length yields a significantly negative slope. of those intervals, contain at least part of an aluy insertion. consistent with the overall pattern, those intervals are longer, on average, than intervals without an aluy insertion. again consistent with the general pattern, the estimated recombination rate in these longer, aluy-containing intervals is lower than in the shorter intervals that lack aluy insertions. the reason for the larger average size of intervals with an aluy in them is unclear. it might be due to the difficulty of designing robust genotyping assays for snps near repeat sequences in the context of a high-throughput genotyping project, or perhaps these repeats are in fact associated with lower nearby genetic variation. we observed a similar pattern with line- insertions. the cause of the general association between estimated recombination rate and inter-snp interval length in hapmap data may be an artifact of the estimation procedure, since regions of lower snp density contain less information about past recombination events. we therefore eliminated the potentially confounding relationship between inter-snp interval size and estimated recombination rate by selecting uniformly sized intervals, re-estimating the recombination rates in those intervals, and also including interval length as a covariate in our regression analyses. previous analyses of correlations between aluy insertions and local recombination rates did not account for this bias and were focused on larger effect sizes, which may explain why no aluy-specific effect was detected. aluy regions in hapmap yri trios to further test the initial results we observed in the african sample of our world diversity panel, we assembled a genome-wide data set of hapmap snps typed on the yoruba parent-child trios. aluy insertions were identified by repeatmasker and excluded known polymorphic insertions by comparison with dbrip. we selected aluy regions and uniformly-spaced snps within them from the hapmap data using an adaptation of the method we used on our world diversity panel above. we analyzed these data after removing the terminal and sub-terminal intervals from each region to eliminate edge effects. table shows the means and standard deviations of log for these intervals and for the seven predictor variables defined for each inter-snp interval: length, regional recombination rate, g+c content, "core" motif count, "extended" motif count, hot spot presence, and aluy presence. after accounting for all other effects in the model, we find that the presence of an aluy insertion predicts a statistically significant though modest increase in recombination rate. the length of an interval has a significant but very small effect on the recombination rate in that interval, which implies that our strategy to eliminate the interval length factor was successful. each of the other variables is independently predictive of the local recombination rate. the impacts of the regional recombination rate and g+c composition are the largest and most significant, as expected. the hot spot-associated recombinogenic motifs are associated with small, local variations in recombination rate independently of their association with hot spots. since those motifs are present in a minority of hot spots, it is not surprising that hot spots themselves have an independent and much stronger effect: an interval that overlaps a known hot spot has a -fold greater recombination rate, on average, compared with nearby intervals that do not overlap a hot spot. aluy regions in hapmap ceu trios with our initial set of aluy regions, we detected no effect of fixed aluy insertions on recombination in our european population sample. we then asked the same question using aluy regions and ceu parent-child trios genotyped by the hapmap project. means and standard deviations of the regression variables are shown in table regression analysis shows that the presence of a fixed aluy insertion is associated with an ~8% increase in the recombination rate in an interval. the overall results are very similar to those observed with the yri data. this similarity is expected, since the patterns of variation and linkage disequilibrium in the ceu population sample are correlated with those in the yri sample because of their shared ancestry. the effect of interval length on recombination rate is small, as it was with the yri data, and statistically insignificant in this case. approximately 16% of the aluy-containing intervals in the ceu data set also overlap a hot spot. while that does not mean that those hot spots overlap the aluy insertions themselves, we nonetheless checked for a potential interaction effect between hot spots and aluy insertions, since such an interaction could account for part of the aluy effect. we observed a significant interaction effect in the ceu hapmap data set. independent effect sizes for aluy and hot spots were slightly but not significantly reduced. both effects remain clearly significant, and the results for other factors are only trivially affected. no significant hot spot × aluy effects were seen in the other data sets. the differences between the results based on our world diversity panel and those obtained from the hapmap data should be considered with caution, due to the small number of regions genotyped in the former. an effect size of ~8% would not be detectable with a sample size of intervals in aluy regions, so the negative results obtained in our european and east asian samples are probably due to a lack of statistical power. although the 95% confidence intervals around the effect sizes estimated for the fixed aluy insertions in the sub-saharan african and the hapmap yri data sets do not overlap, the 99% confidence intervals do. the majority of aluy insertions are 10- million years old. the accumulation of mutations in aluy copies as they age might alter their effects on the local recombination rate. figure explores how the effects of aluy insertions on recombination depend on their degree of sequence divergence from their subfamily consensus sequences, and figure shows the relative frequencies of aluy insertions plotted against their divergence. a similar recombination rate effect is seen across all classes of the aluy divergence spectrum. discussion we have assessed whether aluy insertions affect the recombination rate in their immediate neighborhood. we first generated and analyzed a data set of aluy insertions and surrounding snps that were ascertained to limit extraneous factors and thus to maximize our ability to detect such effects. to test the observations gained from those data, we extended the ascertainment design and analyses to a larger set of aluy insertions and neighboring snps extracted from the hapmap phase ii data. because aluy insertions are correlated with some sequence features that are themselves associated with higher recombination rates or with recombination hot spots, we included those features as covariates in our analyses. we included hot spots themselves as proxies for the as-yet-unknown factors that presumably cause those hot spots. as expected, the average recombination rate within ~ kb on either side of an aluy-containing interval was a strong predictor of that interval's recombination rate. while this yields no insight about the cause of that broad-scale variation, it allows us to factor out any effects at that scale. even with the mean surrounding regional recombination rate already factored out, the g+c content of an inter-snp interval is strongly predictive of its recombination rate. the g+c content itself is correlated with the "core" and "extended" hot spot-associated recombinogenic motifs, since they are gc-rich. nonetheless, both of those motifs carry additional significant predictive power. as expected, the presence of a hot spot in an interval has a much stronger effect, increasing the recombination rate by ~ -fold, on average. there is a slight association between hot spots and aluy insertions: inter-snp intervals that contain an aluy are 13% more likely to overlap a hot spot than control intervals are. some degree of association would be expected under the hypothesis that aluy insertions increase the local recombination rate, since they would push that rate past the threshold for hot spots in at least some regions. there is also some evidence for a positive interaction between hot spots and aluy insertions. however, since many unknown factors may interact to generate recombination hot spots, and since an aluy-specific effect should be detectable independently from those factors and the hot spots they generate, we have attempted to factor out the effect of hot spots. after factoring out effects that are not specific to aluy sequences, we still find that the presence of a fixed aluy insertion has a significant positive impact on the recombination rate within the ~ kb inter-snp interval that contains it. a fixed aluy insertion appears to cause a twofold enhancement of the local recombination rate in the aluy regions we genotyped in our sub-saharan african sample. a smaller positive effect - a % increase over the surrounding intervals, on average - is strongly evident in the larger hapmap-based data sets, for both the yri and ceu populations. no relationship between polymorphic aluy insertions and the local recombination rate was found in the five regions genotyped in our world diversity panel, but a modest effect would not be detectable in a data set of that size. we therefore turned to the hapmap yri trio data set to test for a smaller effect of polymorphic aluy insertions on the local recombination rate. using the methods we applied above to ascertain fixed aluy regions, we identified polymorphic aluy regions based on the aluy loci in dbrip. we examined inter-snp intervals and found no significant effect of the presence of polymorphic aluy elements on local recombination rates. the magnitude of the per-copy effect of a fixed aluy on the local recombination rate is comparable to the effect of the stronger of the two recombinogenic motifs that we analyzed. given the resolution of our data sets, it is possible that the effect may be stronger but more localized than we have reported, since the effect is diluted out over the entire aluy-containing interval. in considering potential causes of the observed effect, it must be noted that the recombination rates estimated here reflect only the history captured by human snps, nearly all of which arose less than myr ago. thus aluy characteristics that existed only prior to that time cannot explain the recent effect of those insertions. aluy sequences might bind cofactors or influence chromatin structure in a way that influences the local recombination rate, as has been suggested for some short recombinogenic motifs. for example, alu insertions are typically flanked on both sides by target sites for line- endonuclease. this is because alu insertions are created by line-1-encoded proteins at line- endonuclease cutting sites, and the original target sites are duplicated during the insertion event. alu insertions may thus attract line- endonuclease, which creates double-strand breaks in the dna that can then be resolved as recombination events. line- endonuclease generates large numbers of dsbs, which suggests that endogenous line- activity might generate dsbs at a rate sufficient to affect recombination rates.  in summary, we have demonstrated that the presence of a fixed aluy element enhances the local recombination rate by approximately 6%. this effect is similar in magnitude to that observed for previously identified recombinogenic motifs. while the effect of each aluy element is relatively small, the presence of hundreds of thousands of these elements throughout the human genome implies that they exert a substantial effect on genome-wide recombination rates. further research is needed to identify precisely the molecular mechanism responsible for this effect. 
to test this observation against a larger set of aluy insertions, we applied our locus- and snp-selection design and analyses to the hapmap phase ii data. recombination rates vary widely across the human genome, but little of that variation is correlated with known dna sequence features. if individual alu insertions have even modest effects on local recombination rates, they could collectively have a significant impact on the pattern of linkage disequilibrium in the human genome and on the evolution of the alu family itself. we carried out sequencing, snp identification, and snp genotyping around aluy insertion loci in individuals sampled from diverse populations, then used the snp genotypes to estimate local recombination rates around the aluy loci. the loci and snps were chosen so as to minimize other factors that could influence recombination rate estimates. we detected a significant increase in recombination rate within ~ kb of the aluy insertions in our african population sample. in that data set, we observed a significantly increased recombination rate near aluy insertions in both the ceu and yri populations. we show that the presence of a fixed aluy insertion is significantly predictive of an elevated local recombination rate within kb of the insertion, independent of other known predictors. the magnitude of this effect, approximately a 6% increase, is comparable to the effects of some recombinogenic dna sequence motifs identified via their association with recombination hot spots.
 recombination rates vary widely across the human genome, but little of that variation is correlated with known dna sequence features. the genome contains more than one million alu mobile element insertions, and these insertions have been implicated in non-homologous recombination, modulation of dna methylation, and transcriptional regulation. if individual alu insertions have even modest effects on local recombination rates, they could collectively have a significant impact on the pattern of linkage disequilibrium in the human genome and on the evolution of the alu family itself.  we carried out sequencing, snp identification, and snp genotyping around aluy insertion loci in individuals sampled from diverse populations, then used the snp genotypes to estimate local recombination rates around the aluy loci. the loci and snps were chosen so as to minimize other factors that could influence recombination rate estimates. we detected a significant increase in recombination rate within ~ kb of the aluy insertions in our african population sample. to test this observation against a larger set of aluy insertions, we applied our locus- and snp-selection design and analyses to the hapmap phase ii data. in that data set, we observed a significantly increased recombination rate near aluy insertions in both the ceu and yri populations.  we show that the presence of a fixed aluy insertion is significantly predictive of an elevated local recombination rate within kb of the insertion, independent of other known predictors. the magnitude of this effect, approximately a 6% increase, is comparable to the effects of some recombinogenic dna sequence motifs identified via their association with recombination hot spots. 