JOURNAL OF COMPUTATIONAL BIOLOGY
Volume 17, Number 9, 2010
# Mary Ann Liebert, Inc.
Pp. 1281–1297
DOI: 10.1089/cmb.2010.0103

An Effective Method for Detecting Gene Conversion
Events in Whole Genomes
CHIH-HAO HSU,1 YU ZHANG,1 ROSS C. HARDISON,1
NISC COMPARATIVE SEQUENCING PROGRAM,2 ERIC D. GREEN,2 and WEBB MILLER1

ABSTRACT
Gene conversion events are often overlooked in analyses of genome evolution. In a conversion event, an interval of DNA sequence (not necessarily containing a gene) overwrites ahighly similar sequence. The event creates relationships among genomic intervals that canconfound attempts to identify orthologs and to transfer functional annotation between genomes. Here we examine 1,616,329 paralogous pairs of mouse genomic intervals, and detect
conversion events in about 7.5% of them. Properties of the putative gene conversions areanalyzed, such as the lengths of the paralogous pairs and the spacing between their sources
and targets. Our approach is illustrated using conversion events in primate CCL geneclusters. Source code for our program is included in the 3SEQ_2D package, which is freely
available at www.bx.psu.edu/miller_lab/.
Key words: algorithms, computational molecular biology, evolution.

1. INTRODUCTION

S

everal classes of evolutionary operations have sculpted genomes. Nucleotide substitutions havebeen studied in great detail for years, and much attention is now focused on large-scale events such as
insertions, deletions, inversions, and duplications. Frequently overlooked are gene conversion events(Hurles, 2004; Chen et al., 2007), in which one region is copied over the location of a highly similar region;
before the operation there are two genomic intervals, say A and B with 95% identity, and afterwards there are
two identical copies of A, one in the position formerly occupied by B.
Conversion events need to be accounted for when attempting to understand the evolution of genomes
based on identification of orthologous regions in other species. To take a hypothetical example, suppose
mouse genes A and B are related by a duplication event that pre-dated the separation of mouse and rat, sothat rat also has genes A and B. A conversion event in a mouse ancestor that overwrote some of B withsequence from A could cause all or part of B’s amino-acid sequence to be more closely related to the rat A
protein than to the rat B protein, even though B’s regulatory regions might remain intact. Successful design
1
Center for Comparative Genomics and Bioinformatics, Pennsylvania State University, University Park,
Pennsylvania.
2
NIH Intramural Sequencing Center (NISC) and Genome Technology Branch, National Human Genome ResearchInstitute, National Institutes of Health, Bethesda, Maryland.

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and interpretation of experiments in rodents to understand gene B might well require knowledge of theseevolutionary relationships.
Gene conversion events have been studied in a variety of species, including the following investigations.
Drouin (2002) characterized conversions within 192 yeast gene families; Semple and Wolfe (1999) detected conversion events in 7,397 Caenorhabditis elegans genes; Ezawa et al. (2006) studied 2,641 gene
quartets, each consisting of two pairs of orthologous genes in mouse and rat, and found that 488 (18%)
appear to have undergone gene conversion; and Xu et al. (2008) detected 377 gene conversion events
within 626 multigene families in the rice genome. However, these studies investigated gene conversion
events only between pairs of protein-coding genes, although conversion can occur between any pair ofhighly similar regions (Chen et al., 2007). Furthermore, these studies only examined a few thousand pairsof genes, while we cover more than one million paralogous pairs of regions, requiring a more efficientmethod to deal with such a large data set.
Evidence of conversion between genes frequently appears in cases where the conversion involves only
part of a duplicated region. For instance, consider the CC chemokine ligand (CCL) gene cluster in primates.
A vervet-vervet alignment reveals similarities extending beyond the genes, created by an ancient duplication event pre-dating the radiation of primates; see Figure 1B. To test whether the elevated percent
identity in the protein-coding regions can be explained entirely by purifying selection on those regions, we
can compare the pattern of sequence conservation between the paralogous vervet regions with that between
vervet CCL15 and its ortholog in an appropriately divergent species. Using dusky titi (a New-World
monkey), we see that in most of the interval around the CCL15 gene, the vervet sequence is more similar tothe dusky titi CCL15 region (thick line) than to the vervet CCL23 region (thin line) as expected, but this isreversed in a large interval containing exons 2 and 3, and also around the region of exon 1; (Fig. 1C). One
reasonable inference from this observation is that conversion events overwrote these intervals with thehomologous sequences from the CCL23 gene, or vice versa. Indeed, our procedure identifies a conversion
event covering an interval that starts somewhat upstream of exon 2 and extends just beyond exon 3. A
smaller interval around exon 1 also shows statistically significant evidence of conversion (Fig. 1D).
A number of statistical tests have been proposed for detecting gene conversions. However, most of theseare only efficient for small data sets, e.g., individual gene clusters. Boni et al. (2007) nicely summarize thecomputational methods available for detecting mosaic structure in sequences, and propose a new method thatis particularly economical in terms of computer execution time for large data sets. One drawback is that theiralgorithm requires large amounts of computer memory. However, we show here that this method can be

FIG. 1. Evidence of gene conversion in the vervet CCL15 gene. (A) Schematic view of the gene. (B) Percent identity
plot of an alignment to an interval containing the vervet CCL23 gene; each short horizontal line indicates the percent
identity over a gap-free subinterval of the alignment. (C) Plot of an alignment to the orthologous dusky titi CCL15 gene(thick line) compared to the same paralogous vervet alignment from the previous panel (thin line). (D) An interval
containing the second and third exons exhibiting gene conversion detected by the method described here, and a smaller
interval around the first exon that also shows statistically significant evidence of conversion. See the text for further
discussion.

GENE CONVERSION EVENTS IN WHOLE GENOMES

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FIG. 2. Timing of evolutionary events. The assumed duplication, speciation, and conversion events between two
species, ie, M and C, occurred respectively x, y, and z years ago so that M1 is orthologous to C1 and M2 is orthologous to
C2. See the text for further explanation.

reformulated so that the memory requirements are no longer a limiting factor, which allows us to conduct acomprehensive scan for gene conversion events across the entire mouse genome, starting with 1,616,329
pairs of paralogous mouse intervals. For each pair of paralogous intervals, say M1 and M2, we choose asequence from another species, say C1, that is believed to be orthologous to M1. These triplets of sequences
are examined to find cases where part of M1 is more similar to M2 than to C1, while another part is moresimilar to C1. In such cases, the interval of high M1  M2 similarity is inferred to have resulted from a
conversion event, as illustrated in Figure 1.
Let us take a closer look at the assumptions implicit in this approach. We are using the sequence C1 in
effect to factor out differences in the rates of evolutionary change along M1. For instance, some positions of
M1 may be evolving neutrally, but at a rate that depends on the adjacent nucleotides (e.g., hypervariable
CpG dinucleotides), while others may be protein-coding or regulatory regions under purifying selection.
For a simple model, denote the expected number of substitutions per year at position p by mp. Also, supposethat this mutation rate holds for all copies after duplication and speciation events, where the duplication
event that separated M1 and M2 happened x years ago, while the speciation event separating M1 and C1
happened y < x years ago (Fig. 2A). Then the expected number of substitutions between (the descendantsof) position p in M1 and M2 is 2xmp, while between M1 and C1 it is 2ymp. The ratio of those two values isx/y > 1, independent of the mutation/fixation rate mp. Thus, in the absence of a conversion event, we expect
M1 to differ from M2 more than from C1 regardless of changes in selective pressure along the sequence.
However, note that changes in selective pressure along the tree branches can produce erroneous signals.
For instance, consider a position that is under strong purifying selection, except on the branch from the M1 /C1 ancestor to C1. Then the total path length from M1 to M2, as weighted by the branch-specific mutation
rates, could be less than that from M1 to C1, which our method would incorrectly interpret as evidence of aconversion event. However, these erroneous signals can be reduced by combining the results from two
triplets. See Methods for a detailed explanation.

2. METHODSBoni et al. (2007) developed a time-efficient method for identifying conversions and other recombination
events, using the M1  M2 and M1  C1 alignments to identify ’’informative’’ positions in M1, such thateither M1 and M2 have one nucleotide while C1 has another (score 1), or M1 and C1 have one nucleotide
while M2 has another (score þ1). The cumulative sum of these scores along M1 constitutes what is called a
hypergeometric random walk (HGRW [Feller, 1957]) under the assumption that M1’s relationships to M2
and C1 are invariant across the interval (Fig. 3). Conversions are detected using the test statistic xm,n,k,which is the probability of a maximum descent of k occurring by chance for a triplet (M1, M2, C1) with m
þ1s and n 1s. The maximum descent is the maximum decrease of scores across the interval, e.g., therectangular regions in Figure 3. Boni et al. (2007) give a dynamic-programming algorithm for computing
xm,n,k, which creates a table that can be consulted for an arbitrary number of triplets.
In order to apply Boni et al.’s method to the entire mouse genome, for each given paralogous pair M1 andM2, we needed to find an orthologous sequence C1 from a species at an appropriate evolutionary distance,i.e., that split from the mouse lineage somewhat after the duplication event and before the conversion. Thus,

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HSU ET AL.

FIG. 3. Maximum descent of the hypergeometric random walks for an alignment between the intervals around thefirst exon of the vervet CCL15 gene and the vervet CCL23 gene. Using the dusky titi sequence as C1 identifies a wider
converted interval than does using gorilla, possibly because of nested conversion events at different times during theevolution of the vervet lineage.

we tried several available mammalian genome sequences: rat, human, and dog. Each of these species canbe used to detect gene conversion events in a particular period of evolution along the lineage leading tomouse. Because the orthologs of M1 and M2 often differ, up to 6 triplets were used to look for gene conversion in a given mouse paralogous pair.

2.1. Space-efficient modifications
The original formulation by Boni et al. (2007) requires an amount of computer time and memory that is
proportional to B4, where B is the maximum of m, n, and k. For a triplet with 400 informative sites, thisapproach would use 6.4 GB of computer memory, allowing the method to work only with relatively shortsequences. We modified that method to need only space proportional to mn þ n2 þ SP (where S ¼ numberof outgroup species and P ¼ number of sequence pairs), as we now describe.
In the notation of Boni et al. (2007), the test statistic xm,n,k is defined as P(md Hm,n ¼ k), which is theprobability for a hypergeometric random walk with m up steps and n down steps, i.e., Hm,n, to havemaximum descent of k, and can be calculated using the equation:
xm, n, k ¼

kXym, n, k, j

(1)

j¼0

where:

\min Hm, n ¼  jym, n, k, j ¼ P md Hm, n ¼ k(2)

The probabilities y, which places one more constriction on the minimum value of Hm,n to be j, can beobtained by dynamic programming based on the following recursive relationships.
8m>if j ¼ 0[ym  1, n, k, 1 þ ym  1, n, k, 0 ]
>
>
m
þ
n
>
>




>
if k > j > 0
< m
n
þ mþ
y
n ym, n  1, k, j  1(3)ym, n, k, j ¼ m þ n m  1, n, k, j þ 1>
if j ¼ k > 0>>n> m þ n [ym, n  1, k  1, j  1 þ ym, n  1, k, j  1 ]>>:if j > k00

GENE CONVERSION EVENTS IN WHOLE GENOMES

1285

With boundary conditions
ym, 0, k, j ¼

y0, n, k, j ¼
ym, n, 0, 0 ¼

1 for k ¼ j ¼ 0
0 otherwise(4)

1 for k ¼ j ¼ n
0 otherwise(5)

1 for n ¼ 0
0 otherwise(6)ym, n, k, j ¼ 0 when k > n or k < n  m:

(7)ym, n, k, j ¼ 0 when j > n or j < n  m:

(8)

Thus, the p-value for a triplet with m þ 1s, n  1s, and maximum descent of k is defined as P(mdHm,n  k) and can be calculated using the following equation:nXxm, n, j(9)p  valuem, n, k ¼
j¼k

In order to reduce memory usage, we introduce the additional variable Am,n,k, defined as:n[Am, n  1, k  1 þ ym, n  1, k, k  1 ]Am, n, k ¼ ym, n, k, k ¼
mþn

(10)

Then,



m
n
ym, n, k, j ¼
xm, n, k ¼
xm  1, n, k þ[xm, n  1, k  Am, n  1, k þ Am, n  1, k  1 ]
mþn
mþn
j¼0
k
X



(11)

The key observation is that, for fixed k, the only component of the equation 3 that depends on k  1 iswhen j ¼ k > 0, and in that case the required value is Am,n  1,k  1. Consequently, provided that we record
the 3-dimensional array of values Am,n,k, we can store the values of y for a fixed k in another 3-dimensional
array that we call ym,n,j and overwrite them with the values corresponding to k þ 1 as the computation
proceeds. The resulting algorithm, given in Figure 4, uses only two arrays of size mn2 (x and y can be stored
in the same array). It can handle triplets with 2000 informative sites on a mid-sized workstation.
Furthermore, since the value of x depends only on the values in the same loop, i.e., xm,n  1,k, and in theprevious loop, i.e., xm  1,n,k (when using m as the outer loop), an O(mn þ n2 þ SP) space method (where
S ¼ number of outgroup species and P ¼ number of paralogous pairs) is possible. Instead of using a three
dimensional array to store all values of xm,n,k, the combinations of (m, n, k) that actually occur in the dataset of
triplets (M1, M2, C1) are determined and stored as nodes in a three-dimensional linked list data structure, as
shown in Figure 5 (triplets with the same values of (m, n, k) are grouped in the same node, and all nodes arelinked in ascending order in three dimensions). This consumes O(SP) space. To obtain the p-values for all
nodes, i.e., equation 9, the values of xm,n,k are calculated and summed to the relevant nodes, i.e., the nodes
between (m, n, 0) and (m, n, k). For this purpose, a two dimensional array called Linked_List_Table, which
points to the starting position for each pair of (m, n), is maintained so that the relevant nodes for a particular
xm,n,k can be retrieved quickly. A detailed algorithm is shown in Figure 6. Since only those values necessary
for further calculation are kept (the values of xm  1,n,k and xm,n,k are stored in the two-dimensional arrays of
x0,n,k and x1,n,k, respectively; x0,n,k and x1,n,k are overwritten with the values corresponding to m and m þ 1 as
the computation proceeds), the maximum table size required for the calculation of x is O(mn þ n2).
Although the space requirement is thus reduced, the time complexity is still quartic (exponent 4). Also, thelongest interval in our data is 394,252 base pairs. In order to deal with long alignments, those with length greaterthan 5000 are divided into several sub-alignments with 1000 sites overlapped. The p-value for each subalignment is then calculated, and a multiple-comparison correction method (Holm, 1979) is used to determine if
the set of sub-alignments supports an assertion that the whole alignment shows significant signs of a conversion.

2.2. Extension to quadruplet testing
It is not uncommon that we have a pair of paralogs in the other species, say C1 and C2 in rat, that areorthologs for M1 and M2 in mouse, respectively. In a fashion similar to the triplet testing, we can perform

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HSU ET AL.

FIG. 4.

A cubic-space algorithm for computing the probabilities xm,n,k.

quadruplet testing (M1, M2, C1, C2) that is the summation of the hypergeometric random walks of two
triplets, i.e., (M1, M2, C1) and (M1, M2, C2), as shown in Figure 7. This joint testing of gene conversions
often has higher power than triplet testing. For example, in a four-way alignment if we observe thatM1i ¼ M2i = C1i ¼ C2i at a column i, this is strong evidence for gene conversion.
We again need to assign a score to each column in the four-way alignment in order to calculate
maximum descent scores. We use the sum of the triplet scores. In the quadruplet case of (M1, M2, C1, C2),
there are two distinct triplets of interest: (M1, M2, C1) and (M1, M2, C2). Other possible triplets are ignored,
as we are testing for gene conversions in mouse. In the case of M1i ¼ M2i = C1i ¼ C2i, we assign a score of
2 to column i, because each triplet (M1i ¼ M2i = C1i and M1i ¼ M2i = C2i) has score 1. On the other
hand, if M1i ¼ C1i = M2i ¼ C2i, we assign a score of þ2 by the same rule. Furthermore, if M1i = M2i ¼
C1i ¼ C2i or M2i = M1i ¼ C1i ¼ C2i, we assign score þ1 because one triplet has score þ1 and the other hasscore 0 (non-informative). In summary, all columns are assigned the sum of their two triplet scores, and allcolumns with 0 score are subsequently ignored.
Suppose that there are m1 one-step-ups, m2 two-step-ups, n1 one-step-downs, and n2 two-step-downs fora particular quadruplet testing. Let N ¼ m1 þ m2 þ n1 þ n2 denote the total number of moves. We calculate
the exact p-value of observing at least k maximum descent in a random walk, constrained by (m1, m2, n1,n2) moves of each type, using the following recursive formula:
xm1 , m1 , n1 , n2 , k ¼

k
X
j¼0

ym1 , m1 , n1 , n2 , k, j

(12)

GENE CONVERSION EVENTS IN WHOLE GENOMESFIG. 5.

A 3-D linked list data structure to store necessary values for computing the probabilities xm,n,k.

FIG. 6.

A quadratic-space algorithm for computing the probabilities xm,n,k.

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HSU ET AL.

FIG. 7. Comparisons between quadruplet testing and triplet testing. (A) The colobus monkey CCL3 and a partial
pseudo gene paralog pair. (B) The vervet CCL15 and vervet CCL23 paralog pair.

where
8 m P1m2 P2
1>l ¼ 0 ym1  1, m2 , n1 , n2 , k, l þ N
l ¼ 0 ym1 , m2  1, n1 , n2 , k, lN>>> m1 ym  1, m , n , n , k, j þ 1 þ m2 ym , m  1, n , n , k, j þ 2 þ
<
1
2 1 2
1
2
1 2
N
N
ym1 , m1 , n1 , n2 , k, j ¼ nN1 ym1 , m2 , n1  1, n2 , k, j  1 þ nN2 ym1 , m2 , n1 , n2  1, k, j  2>>
n 1 Pkn2 Pk>>: N l ¼ k  1 ym1 , m2 , n1  1, n2 , l, j  1 þ N l ¼ k  2 ym1 , m2 , n1 , n2  1, l, j  20

if j ¼ 0
if k 4 j 4 0
if j ¼ k 4 0
if j 4 k  0(13)

The y terms are set to 0 if their subscripts go below 0 or above k. Let n ¼ n1þ2n2 and m ¼ m1þ2m2, thenk is bounded between [max(n  m, 1), n] for m > 0, n > 0, and the computation time for the p-values of all
possible (m1, m2, n1, n2, k) combinations is O(m1m2n1n2n2), and the memory usage is O(m1m2n1n2n).
However, since the time complexity and memory consumption for this formula are very high in practice,
we use the same formula as in triplet testing, i.e., equation 11, to get p-values in our program, even though
it is more conservative.
Quadruplet testing often has higher specificity and sensitivity than triplet testing for detecting conversions. For example, in Figure 7A, a weakly significant (0.012) conversion event in colobus monkey was
detected between CCL3 and a partial pseudo gene in one triplet, but there is no evidence for the event in theother triplet. This could be due to a faster evolutionary rate in the (probably non-functional) partial genethan in the coding region of CCL3. Quadruplet testing did not show any evidence of conversion in thisregion, which suggests that the effect of one triplet can be neutralized by that of the other triplet when there
is no conversion between a paralog pair. On the other hand, in cases where the triplets reinforce each other,quadruplet testing can give a more significant result, as shown in Figure 7B. Therefore, whenever orthologs
for both M1 and M2 are available in a particular outgroup species, we combine the results of the two triplets
to perform quadruplet testing.

2.3. Multiple-comparison correction
When several statistical tests are performed simultaneously, a multiple-comparison correction should
be applied. In our study, three outgroup species are used, and for this multiplicity we use the Bonferroni
correction (Holm, 1979); we multiply the smallest p-value for each paralogous mouse pair by the number oftests (up to 3), and use this adjusted p-value to evaluate the significance of any potential gene conversion inthat pair.
Multiple-comparison correction is also applied to compensate for the many pairs of paralogous sequences. For the 1,616,329 pairs that were analyzed, we used a correction method that controls the false
discovery rate (FDR), proposed by Benjamini and Hochberg (1995). The cutoff threshold for p-values canbe found by the following algorithm:

GENE CONVERSION EVENTS IN WHOLE GENOMES
Algorithm.

1289

CutOff(a, p-values)

1 sort p-values in ascending order
2 for i / 1 to number of p-values do
3
if pi > (i 7 number of p-values)a4
return (i 7 number of p-values)a

In our mouse study, a was set to 0.05 and the corresponding cutoff threshold for p-values was 0.003771.
This means that only a test whose p-value after Bonferroni correction was less than 0.003771 was considered as significant evidence for gene conversion.

2.4. Directionality of gene conversion
We attempt to determine the source and target of a conversion event as follows. As shown in Figure 2B, let
us suppose that duplication, speciation, and conversion events occurred x, y, and z years ago respectively,
with x > y > z, and consider a converted position. Regardless of the direction of the conversion (from M1 to
M2, or vice versa), M1 and M2 are separated by 2z total years in the converted region. If M1 converted M2 (i.e.,part of M1 overwrote part of M2), then the separation of M1 and C1 is 2y but the separation of M2 and its
ortholog, C2, is 2x > 2y. This observation serves as a basis for determining the conversion direction. Figure 8
shows an example of determining the source and target of a conversion from CCL23 to CCL15 in vervet.
Specifically, assume (m1, n1) with maximum descent k1 in the first triplet (M1, M2, C1), and (m2, n2) withmaximum descent k2 in the second triplet (M1, M2, C2). Note that mi and ni here are not the m and n inequation 1; rather, they are the numbers of ups and downs within the common maximum descent regions of
the two triplets (intersection). The probabilities of going down in these regions are:p1 ¼ n1  (m1 þ n1 )p2 ¼ n2  (m2 þ n2 )

(14)
(15)

When combining these data in a quadruplet, there are a total of (m1 þ m2) ups and (n1 þ n2) downs, andthe possibility of going down in the combined data is:
p ¼ (n1 þ n2 )  (m1 þ m2 þ n1 þ n2 )(16)

As shown in Figure 2B, if M1 converted M2, the separation of M1 and C1 is closer than the separation of M2
and C2 in the converted region. Thus, p1 should be smaller than p2. Our objective function (O) is therefore
to determine how significant the difference of p1  p2 is, based on the binomial distribution:

FIG. 8. Evidence that the vervet CCL23 gene converted the vervet CCL15 gene. Percent identity plots for (A) CCL15
and (B) CCL23 showing alignments to the vervet paralog as a thin line, and alignments to the putative colobus ortholog
as a thick line. In the converted region, the vervet-colobus alignments have 94% identity for CCL15 and 97% identity
for CCL23.

1290

HSU ET AL.
O ¼ ((p1  p2 )  E(p01  p02 ))  sqrt(V(p01  p02 ))(17)

E(p01  p02 ) ¼ 0


1
1
þ
V(p01  p02 ) ¼· p · (1  p)
m1 þ n1 m2 þ n2

(18)

where

(19)

In our study, three outgroup species are used to detect gene conversions in the mouse genome. We use
the species that shows the most significant difference of p1  p2 to determine the directionality of conversion for a given paralogous pair. However, there are several reasons why the direction of a conversion
might not be clear, even when using several outgroup species, such as conversions in the outgroup species
or missing outgroup data. Only part of the direction for the putative conversions can be determined.

3. RESULTS
3.1. Highly conserved pairs of sequences
We aligned each pair of mouse chromosomes that are masked out with REPEATMASKER (Smit, 1999),
including self-alignments, using BLASTZ (Schwartz et al., 2003) with T ¼ 2 and default values for theother parameters. Alignments with identity of less than 70% were removed. Chaining of the mouse-mousealignments was performed using the method of Zhang et al. (1994). For alignments between mouseintervals and their putative orthologs in other species, we used the pairwise alignment nets (Kent et al.,2003) downloaded from the UCSC Genome Browser website (Kent et al., 2002). We applied the modified
method described in this paper and recorded information about the inferred conversion events in Table 1.
Of the 1,616,329 analyzed pairs of mouse sequences, 121,899 (7.5%) indicated a gene conversion event.
The fraction of intra-chromosomal pairs indicating a conversion (13.8%) is significantly higher than forinter-chromosomal pairs (6.7%).

3.2. Association with gene conversion
To study the correlations between various genomic features and gene conversion, we used logistic
regression models (Agresti, 2002) to characterize gene conversions based on the following factors.
strand:seq_len:pair_dist:
seq_sim:
gc:
gc1:
gc2:
coding1:
coding2:

binary; strand of the second paralog relative to the first one
paralog size in basepairs
distance between the paralogs
percent identity between the two paralogs
percentage of G þ C in both paralogs combined
percentage of G þ C in the first paralog
percentage of G þ C in the second paralog
binary; whether or not the first paralog contains coding regions
binary; whether or not the second paralog contains coding regions

To make the analysis robust, we first binned the continuous factors into ordered categories shown as
Table 2.
For ease of interpretation, we only included the main effects of the variables, and carried out the analysis
separately for inter-chromosome and intra-chromosome pairs, as shown below under (1) and (2), respectively. For a logistic regression model, the response is binary. In our case, it is gene conversion (indicated
Table 1. Distribution of Intra- and Inter-Chromosomal Gene Conversions

Gene conversion
No gene conversion
Total

Intra-chromosome

Inter-chromosome

Total

25,189 (13.8%)
157,833 (86.2%)
183,022

96,710 (6.7%)
1,336,597 (93.3%)
1,433,307

121,899 (7.5%)
1,494,430 (92.5%)
1,616,329

GENE CONVERSION EVENTS IN WHOLE GENOMESTable 2.

1291

Categories for Continuous Paralog Properties

Categories

0

1

2

3

4

5

seq_lenpair_dist
seq_simgc, gc1, gc2

<200
inter
<0.75
<0.4

200–500<1k
0.75–0.8
0.4–0.45

500-1k
1k–10k
0.8–0.85
0.45–0.5

1k–2k
10k–100k
0.85–0.9
0.5–0.55

2k–5k
100k–1m
0.9–0.95
0.55–0.6

5k
1m–10m
0.95–1
0.6

6

7

10m–100m

100m

by 1) or not (indicated by 0). The logistic regression relates the logit of the probability of gene conversion
to a function of predictors, where the logit function is logit(x) ¼ log(x 7 (1  x)). The actual gene conversion event is regarded as a binary outcome with the probability given by the regression model.
(1) For inter-chromosome paralog pairs, the model is:
logit(conversion_rate) *seq_len þ seq_sim þ gc þ coding1 þ coding2
Coefficients:(Intercept)
seq_len
seq_simgc
coding1
coding2

Estimate

Std. errorz Value

Pr(>jzj)3.598053
0.730605
0.114288
0.050462
0.122936
0.361988

0.008202
0.003035
0.002934
0.003065
0.021215
0.020886438.668
240.759
38.95916.462
5.795
17.332< 2e-16***< 2e-16***< 2e-16***<2e-16***6.84e-09***< 2e-16***

If the estimated coefficient of a variable is positive, the variable increases the gene conversion probability (or rate), while a negative value indicates a decrease.
(2) For intra-chromosome paralog pairs, we included two more variables, strand and pair_dist, the modelis:logit(conversion_rate) *strand þ seq_len þ seq_sim þ gc þ coding1 þ coding2 þ pair_dist
Coefficients:(Intercept)
strandseq_len
seq_simgc
coding1
coding2pair_dist

Estimate

Std. errorz Value

Pr(>jzj)

1.719952
0.032592
0.520803
0.030534
0.158865
0.277185
0.1933120.269120

0.030422
0.014835
0.004999
0.006159
0.006520
0.023922
0.024272
0.005643

56.536
2.197
104.172
4.958
24.365
11.5877.964
47.688< 2e-16***0.028*< 2e-16***
7.12e-07***< 2e-16***< 2e-16***
1.66e-15***< 2e-16***

Based on the results of (1) and (2), we see that:
The conversion rate is higher when M1 and M2 are on the same chromosome; this can be seen from thelarger intercept of (2) than of (1).
 Strand has little effect on gene conversion. It seems natural that relative strand is not a factor when theparalogs are on different chromosomes. When M1 and M2 are on the same chromosome, from (2) we
see that strand effect is positive (0.028) but only weakly significant.
 Conversion rate increases as paralog size increases; this can be seen from the positive coefficient of
seq_len in both (1) and (2).
 Similarity of sequences has a significant effect on conversion rate for both inter- and intra-chromosome
pairs.


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Both GC content and the presence of coding sequences contribute positively to the conversion rate forinter- and intra-chromosome pairs.
 Conversion rate decreases as the distance between two paralogs increases; this can be seen from (2)where the coefficient of pair_dist is negative.
The models used here are simplified; they do not account for interactions among factors. We did notinclude interactions because (a) they are more complicated to interpret, and (b) they would require much
more computer memory, considering that over a million paralog pairs were being tested. Instead, we
performed small-scale studies using subsets of the data, and we observed that although some interactions
are significantly related to gene conversion rate, the magnitude of their contributions is relatively small
compared to the factors direct effects. Furthermore, we did not account for differences among chromosomes, although conversion rates do vary significantly depending on the chromosome.

3.3. Directionality of gene conversion
To obtain a logistic regression model for the conversion direction (in the cases where it could be determined), we used the discrete variable con_direction, set to 1 if H2 converts H1, and 0 if H1 converts H2.
(1) For inter-chromosome paralog pairs, the model is:
logit(con_direction) *seq_len þ seq_sim þ gc1 þ gc2 þ coding1 þ coding2
Coefficients:(Intercept)
seq_len
seq_sim
gc1
gc2
coding1
coding2

Estimate

Std. errorz Value

Pr(>jzj)

0.0098630
0.0001498
0.0120501
0.6115270
0.6031434
1.1629914
1.2867614

0.0178540
0.0074786
0.0062416
0.0109130
0.0108527
0.0487090
0.0481985

0.552
0.0201.931
56.037
55.576
23.876
26.6970.58070.98400.0535< 2e-16***< 2e-16***< 2e-16***< 2e-16***(2) For intra-chromosome paralog pairs, the model is:
logit(conversion_rate) *strand þ seq_len þ seq_sim þ gc1 þ gc2 þ coding1 þ coding2 þ pair_dist
Coefficients:(Intercept)
strandseq_len
seq_sim
gc1
gc2
coding1
coding2pair_dist

Estimate

Std. errorz Value

Pr(>jzj)

0.213053
0.096799
0.017195
0.003534
0.604822
0.6122930.568145
0.398675
0.029474

0.072980
0.031290
0.011786
0.013072
0.028992
0.028947
0.049264
0.0491200.013091

2.919
3.094
1.459
0.27020.862
21.152
11.533
8.116
2.2510.00351**0.00198**
0.14461
0.78691< 2e-16***< 2e-16***< 2e-16***
4.8e-16***0.02435*

Based on the results of (1) and (2), we see that:
Sequence length and similarity are not significantly associated with conversion direction.
Strand and pair distance have slight effects on conversion direction.
 GC content and coding sequences significantly affect the direction for both inter- and intrachromosome pairs. Negative gc1 and positive gc2 suggest that the conversion direction is more likely
to be from the region with higher GC-content to the one with lower GC content. Negative coding1 andpositive coding2 mean that the conversion direction tends to be from a functional gene to a pseudogene.
GENE CONVERSION EVENTS IN WHOLE GENOMES

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3.4. Analysis of CCL gene cluster (hg18.chr17:31,334,806-31,886,998)
In order to evaluate the performance of our method, we tested it on the CCL gene cluster in sevenprimates. We searched for conversion events in each of the species, using the other six as outgroups. We
also compared our results to those from another gene conversion detection method, GARD (Pond et al.,2006), for confirmation.
The CCL gene cluster contains several chemokine ligand genes, some of which affect the ability of the
HIV virus to enter the cell (Modi et al., 2006). In our analysis, we found that this gene cluster expanded
after the separation of humans and Old World monkeys, and detected a number of conversion events shownin Figure 9. Within the gene-cluster sequences of the seven primates, we identified 42 genes and 5 pseudogenes using GeneWise2 (Birney et al., 2004). Our results show that several conversion events occurred
between CCL15 and CCL23 in different lineages. In addition, a number of gene conversion events weredetected in non-coding regions.
Based on our results, we found that several conversion events occurred involving the first exon in the
CCL15 and CCL23 genes along various species. Also, an earlier conversion event occurred in the region
containing the second and third exons followed by a small conversion event around the second exon; there isno conversion in the fourth exon. To show the correctness of our results, we separated the alignment of
CCL15 and CCL23 genes into four regions as shown in Figure 10A and constructed a phylogenetic tree foreach region using the PHYLIP package (Felsenstein, 1989). The phylogenetic trees are shown in Figure 10B–E
and they are different from each other. Also, we used the GARD program to confirm this result. The GARD
program reports evidence of three breakpoints, shown in Figure 11; they almost separate the whole alignment
into four regions near the boundaries of exons. Both of these two results indicate that the evolutionary
histories of these four regions are different. Our results can give a reasonable explanation about the inconsistencies among these four phylogenetic trees. The CCL15 and CCL23 genes were separated by an ancient
duplication event pre-dating the radiation of primates. Several gene conversion events occurred in the first
three exons after the split of simian primates and lemurs. Following the separation of New World and Old
World monkeys, a conversion event occurred in Old World monkeys covering an interval that starts
somewhat upstream of exon 2 and extends just beyond exon 3, and another one occurred in the region
containing exon 1. Finally, after the separation of apes and Old World monkeys, there was a conversion eventin the region containing exon 2 in the human lineage, and another in the vicinity of exon 1 in the Old World
monkeys. The inferred evolutionary history of the primate CCL15 and CCL23 is shown in Figure 12.
Therefore, combining this with our previous studies, a possible evolutionary history for the entire CCL
gene cluster is shown in Figure 13. There were six genes in the root node (primates), and the cluster has
undergone significant expansion after the separation of human and Old World monkeys. Several geneconversion events occurred along various lineages.

FIG. 9. Gene conversion events detected in the primate CCL gene cluster. Arrows show the directionality of
conversion. Several gene conversion events occurred between the coding sequences of the CCL15 and CCL23 genes.
Also, a number of conversion events occurred in the non-coding regions or between the flanking non-coding sequenceand intron, i.e., around the CCL14 gene of Ag Monkey.

FIG. 10. Phylogenetic trees for four regions within the CCL15 and CCL23 genes in the primate CCL gene cluster.
(A) Aligned sequences from the cluster, where only informative sites are shown. The alignment is divided into four
regions based on our detected conversion events. (B–E) Maximum-likelihood phylogenetic trees (1000 bootstraps) areconstructed for the regions containing exons 1–4, respectively.

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FIG. 11. Results from the GARD program (Pond et al., 2006). Evidence for three breakpoints is found, dividing thewhole alignment into four regions near the boundaries of exons.

4. DISCUSSION
For much of the half-century since multi-gene families were discovered, it has been known that copies of
the repeated genes within a species are often more similar than would be expected from their interspecies
divergence. The processes generating this sequence homogeneity in repeated DNA are mechanisms ofconcerted evolution. Gene conversion is one of these processes, and while its impact on disease genes isappreciated (Chen et al., 2007), the extent of its impact on the evolution of the mouse genome has not been
fully investigated in previous studies. Our work documents about one hundred and fifty thousand conversions (7.5%) between duplicated DNA segments in mouse. Similarly large fractions of conversion
events among duplicated segments have been reported in whole-genome studies of yeast (Drouin, 2002),
Drosophila melanogaster (Osada and Innan, 2008) and rodents (Ezawa et al., 2006), though the total
number of observed gene conversions is much higher in our study. The genome-wide identification of DNA
segments undergoing concerted evolution via gene conversions will make the application of comparative
genomics to functional annotation considerably more accurate. This resource will allow the conversion

FIG. 12. Inferred evolutionary history of the primate CCL15 and CCL23 genes. Four different species, i.e., black
lemur (B), howler monkey (A), colobus monkey (C), and human (H), are shown in these trees.

1296

HSU ET AL.

FIG. 13. Inferred evolutionary history of the CCL gene cluster. Here, superscript p indicates a pseudo-gene and ’’-’’
indicates a gene on the reverse strand.

process to be factored into functional inference based on sequence similarity to other species; for example,
it could flag potential false positives for inferred positive or negative selection.
We also examined the association of gene conversion with various genomic features. In particular, wefind that the length of paralogous segments has a strong positive effect on conversion events for both interand intra-chromosomal paralog pairs, as expected for a process requiring homologous pairing. Also, twofindings indicate that closer proximity between the homologous pairs increases the likelihood of a conversion event: the conversion frequency is higher for intra-chromosomal pairs than for inter-chromosomal
ones, and it is also higher for paralog pairs that are closer together on a chromosome. The closer proximity
may be expected to increase the frequency of homologous pairing in recombination. Furthermore, theeffects of coding sequences are very interesting. They have a positive correlation with conversion events,
which could result from higher similarity. Also there is a more frequent conversion direction from afunctional gene to a very similar pseudogene than vice versa, which could be a consequence of selection.
The correlation with sequence similarity and the effects of GC content provide additional information about
the occurrence of gene conversion events.

ACKNOWLEDGMENTS
This work was supported by grant HG02238 from the National Human Genome Research Institute toW.M., grant DK065806 from the National Institute of Diabetes, Digestive and Kidney Diseases to R.C.H.,
and funds provided by the Intramural Research Program of the National Human Genome Research Institute. We thank Maciek Boni for explaining his strategies for reducing the computer memory required byhis method.

GENE CONVERSION EVENTS IN WHOLE GENOMES1297

DISCLOSURE STATEMENT
No competing financial interests exist.

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Address correspondence to:
Dr. Chih-Hao Hsu
Center for Comparative Genomics and Bioinformatics
Pennsylvania State University
University Park, PA 16802
E-mail: cxh503@psu.edu