BACKGROUND
african clawed frogs comprise more than twenty species of frogs native to sub-saharan africa  <cit> .
the most studied species in this genus are xenopus laevis and more recently xenopus tropicalis.
xenopus species have been an important model in cell biology, development, genetics and genomics.
these species are an attractive model in these areas based on the ability to study embryos at all developmental stages, the presence of large eggs in abundant quantities throughout the year and the remarkable regenerative capacity in the tadpole.
xenopus research has set key principles in gene regulation and signal transduction, embryonic induction, morphogenesis and patterning as well as cell cycle regulation  <cit> .
historically, x. laevis has been considered one of the main animal models for developmental, cell, electrophysiology and biomedical studies .
however, this species presents a challenge for genomics analyses and genetics due to the allotetraploid nature of its genome and its long life cycle.
the haploid genome of x. laevis has been sequenced to  <dig>  % and consists of  <dig> chromosomes and  <dig> gbp .
current assembly of the x. laevis genome consists in  <dig>  scaffolds in the xenbase release  <dig>    <cit> .
this release includes the identification of l  and s  chromosomes from the new nomenclature by matsuda et.
al.
<cit> .
the x. laevis transcriptome counts with  <dig>  primary transcript sequences.
the annotation of the transcripts, in the current release, include the identification of the genes known to be duplicated, that belong to chromosomes l and s  <cit> .
one limitation of x. laevis, however, has been the lack of systematic genetic studies to complement molecular and cell biology investigations.
work with the closely related diploid frog x. tropicalis has attempted to address this limitation  <cit> .
x. tropicalis  is a diploid organism with  <dig> chromosomes and a  <dig> gbp long haploid genome.
currently,  <dig>  % of the genome has been sequenced, consisting of  <dig>  scaffolds in xenbase release  <dig>  .
the first and longest  <dig> scaffolds correspond to  <dig>  % of contiguous sequences of the  <dig> haploid chromosomes in the x. tropicalis genome.
this organism has  <dig>  transcript sequences .
the easy molecular tractability of genomic features of x. tropicalis  <cit>  has allowed integration of some genetic, biochemical, phenotypic and evolutionary data  in these two species.
however, correspondence is not always expected between genomic data in x. tropicalis and the duplicated and divergent genome of x. laevis  <cit> .
in the case there is correspondence, establishing it at a genome level is required.
this cannot be done without a physical map between both genomes.
no comprehensive comparative analyses using genomic sequencing mapping have been conducted for x. laevis and x. tropicalis  <cit> .
aiming at facilitating such analysis, we have set out to build a comparative coarse-grained physical map between these two species.
to this end, we aligned the  <dig> chromosomes from x. laevis assembly xla <dig>  to the  <dig> chromosomes from x. tropicalis assembly xtr <dig>  and estimated percentage of sequence identity, repetitions, inversions and synteny of mapped genes between the two species.
finally, we validated the map theoretically through the synteny of maximal unique matches .
as a whole, our results convey the suitability of this newly assembled map for comparative studies between these two species, bridging a long-standing gap for the integration of biochemical, genetic and genomics data in xenopus.
RESULTS
in this work we have performed a comparative analysis between the two frog genomes after mapping by a coarse-grain alignment method the chromosome sequences of x. laevis on the chromosome sequences from x. tropicalis and semi automatic annotation of their transcripts  to complement the map information.
the analyses include a validation of the map, estimations of percentage of sequence identity, repetitions, inversions and synteny between the two genomes.fig.
<dig> a chart summarizing the workflow from the two xenopus assemblies to the map and the analyses
the map
as x. laevis genome is around  <dig>  times the length of x. tropicalis genome,  <dig>  is also the expected rate of added lengths of the blocks aligned between the two species.
this rate depends on the alignment drop-off score, x, chosen.
a resulting rate larger than  <dig>  suggests a loose alignment.
on the other hand, a resulting rate smaller than  <dig>  suggests a strict alignment.
the drop-off score x =  <dig>  rendered an average alignment length rate of  <dig> , which is close to the expected rate .
however, the rate between the lengths of the chromosomes from x. laevis respect to x. tropicalis is  <dig> , larger than expected.table  <dig> summary of the coarse-grained map between  <dig> xla <dig>  chromosomes  on  <dig> xtr <dig>  chromosomes.
the length units are in blocks.
each block corresponds to a sequence of length  <dig> kbp.
xtr ; xla ; chr
ain x. laevis, chromosomes  <dig> and  <dig> from x. tropicalis become fused and duplicated.
they were named chr9_10l and chr9_10s chromosomes in xla <dig> .
the same set was aligned to x. tropicalis chromosome  <dig> and chromosome 10
a coarse-grained dotplot alignment between x. laevis scaffolds and each x. tropicalis chromosome scaffold shows graphically part of the information in table  <dig> .
although the alignments seem to be contiguous, overall  <dig>  % of x. tropicalis chromosomes did not align to x. laevis chromosomes.
in supplement to this figure, the proportion of x. tropicalis chromosomes covered by x. laevis was  <dig>  % .
this proportion, combined with the completion of  <dig>  % of the x. tropicalis genome , results that  <dig>  % of x. tropicalis whole genome is actually aligned by x. laevis blocks.
a similar coverage of  <dig>  % was obtained for x. laevis chromosomes .fig.
<dig> dotplot alignments of each xla <dig>  l and s chromosomes  to each xtr <dig>  chromosome .
a red dot represents a block alignment between x. laevis and x. tropicalis chromosomes.
the alignments are not at the same scale
conservation between x. tropicalis and x. laevis
as the resulting alignment depends on the drop-off value used, we aligned all x. laevis scaffolds against all x. tropicalis chromosomes at  <dig> increasing drop-off score values  .
the block positions that appear with no conservation are either not aligned or have a score lower than  <dig> , in which case cannot be distinguished from chance.
the maximum drop-off score at which a pair of blocks can be aligned correlates directly with percentage of sequence identity between aligned sequences.
however, as the variance of the percentage of sequence identity per drop-off score value is significant, the percentage of sequence identity cannot be reliably predicted from the drop-off score.
in spite of this, the maximum drop-off score at which a pair of blocks is aligned can be used as a measure of conservation.
from each chromosome, a histogram of maximum drop-off scores or conservation scores was generated and the coverage of alignment for each drop-off was calculated.
the average maximum cgaln drop-off score between the aligning zones of the genomes is  <dig> .
<dig> .
possibly, the histogram of maximum drop-off scores shows a larger than expected proportion of conserved blocks with score of  <dig> , as that bin accumulates all blocks with drop-off score  <dig>  or higher.
chromosome  <dig> is the shortest chromosome, and the one that has the lowest average conservation  and lowest alignment coverage .
in order, from highest to lowest average conservation we have x. tropicalis chromosomes:  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> .
this chromosome conservation order changes to  <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig>   <dig> and  <dig> if the averaging only takes into account the aligning blocks.fig.
<dig> maximum drop-off scores chart of x. laevis blocks on x. tropicalis.
a bar-plot of the maximum drop-off score, x, per block position for chromosome  <dig>  b histogram of drop-off scores.
c table with the average maximum drop-off scores per chromosome calculated using all blocks, only the aligning blocks, and the coverage of the aligning blocks on x. tropicalis.
the cells in grey show the most conserved chromosomes according each column
repetitions and inversions
as x. laevis genome is the result of whole genome duplication event, it is expected that  <dig>  x. laevis blocks will align each x. tropicalis block.
therefore, a block of nucleotides cannot simply be regarded as a block that happens more than once in a genome.
three particular cases have to be taken in account: a block from x. tropicalis that aligns to x. laevis is considered a repeat when  it is an additional block to an already-aligned first block at one particular scaffold;  it belongs to a third scaffold in addition to two previous aligned scaffolds or;  it is a combination of the former two cases.
in this map, a total of  <dig> mbp from x. tropicalis are repeated in  <dig> mbp in the x. laevis aligned genome .
inversions are identified only for colonies, i.e., with at least two consecutive aligning blocks  <cit> .
for colonies, a previous check on the scaffold frame is made, as in cgaln only the best out of the  <dig> reading frames of each x. laevis scaffold is aligned.
an inversion is identified when cgaln takes the plus frame of the x. laevis chromosome and a colony is aligned in reverse respect to the x. tropicalis chromosome.
because only colonies in reverse can be identified, the inversions counted are an underestimation of the total number of existing inversions.
taking into account this limitation, we estimated at least  <dig> mbp to be inverted between the two genomes.
inversions represent 7 % and 3 % of the aligned portion of x. tropicalis or x. laevis genomes, respectively .table  <dig> summary of repetitions  and inversions in the coarse-grained map between  <dig> xla <dig>  chromosomes on  <dig> xtr <dig>  chromosomes.
columns  <dig> to  <dig> are sub estimates of the number of repeated blocks from each genome that align on the other genome.
columns  <dig> to  <dig> are sub estimates of inversions between the genomes
validation of the map
in order to validate the map between x. laevis and x. tropicalis, we computed a set of common theoretical probes called maximal unique matches  between the two genomes and compared their correlative order in the map.
the mums generated were identical between species and 250 nt or longer.
the distribution of distances between the corresponding positions in the map for the mums gives a measure of how well the correspondence between the genomes was achieved.
the generated list of mums has  <dig>  sequences.
from those,  <dig>  were mapped on the ten x. tropicalis chromosomes and  <dig> were mapped on the x. laevis scaffolds;  <dig> mums, representing  <dig>  % of the total, are common and mapped to both species.
this number is less than expected as it is lower than the proportion of the x. laevis genome mapped.
additionally,  <dig>  or  <dig>  % of the mapped mums on x. laevis are at a distance of ≤5kbp from the corresponding mum in x. tropicalis.
one block, or 5kbp, is the resolution of the map.
therefore, we estimate that the correspondence between the two sets of scaffolds was achieved in  <dig>  % of the map.
application of the map: conserved synteny and gene rearrangements
to calculate conserved synteny, a set of orthologous genes between two species is required.
<dig>  orthologous genes were found through bidirectional-best-hit using blastn.
a subset of these,  <dig>  genes, map on the x. tropicalis  <dig> chromosomes.
out of all x. laevis transcripts, only  <dig>  map on x. tropicalis chromosomes .
from these,  <dig>  are orthologous genes and present in at least pairs of consecutive orthologous genes mapped in the same x. laevis chromosome.
this set was our orthologous genes sample for synteny estimation.
we found that  <dig>  orthologous genes, or  <dig>  % of the sample, are syntenic between the two species.table  <dig> distribution of xla <dig>  transcripts according to its mapping on xtr <dig>  chromosomes assembly.
a transcript is considered partially aligned if only one of the blocks, either the one including the start or the stop position, is aligned.
a transcript does not align on x. tropicalis if neither of the blocks that include start or stop positions, is aligned
because the intergenic distance is one of the main determinants of order conservation  <cit> , three distances were measured between pairs of orthologous genes : 1) distance between two consecutive genes in x. laevis; 2) distance between two consecutive genes in x. tropicalis and; 3) distance between x. laevis start block position projected on x. tropicalis and its orthologous gene start block.fig.
<dig> three distances measured between the consecutive genes in x. laevis , a and b as intergenic-distance, igd; between two consecutive genes in x. tropicalis , a’ and b’ as igd and; the distance between orthologous genes in both genomes d
the relative error of the distance between two consecutive genes in x. laevis respect to x. tropicalis was calculated with the first two distances.
the mean relative error was  <dig>  %.
this means that regardless the absolute distance between two consecutive orthologous genes in x. tropicalis, the corresponding consecutive genes in x. laevis are, in average, ±  <dig>  % of that distance apart.
<dig>  % of the orthologous pairs of genes are in the corresponding block position according to the map.
in the case of the distribution for the third measured distance, it was found that orthologous genes are mapped, in average 9kbp, and that 95 % of the orthologous genes are at most 55kbp apart.
for comparison, the confidence interval of lengths, at 95 %, of xenopus genes are between  <dig> and 15kbp.
percentage sequence identity between the two species
based on the calculated mapping between the two species, and to assess more precisely the sequence conservation, a random sample containing 100mbp of matching blocks were aligned by using the global needleman-wunsch and local smith-waterman dynamic programming algorithms.
the aim was to estimate, respectively, upper and lower references of the sequence identity between the two xenopus species.
for the two types of alignments, median percentage sequence identities are similar, both per chromosome and in total .
the distributions for global and local alignment overlap .
the medians are  <dig>  and 43 %, respectively.
in average, the percentage sequence identity shared by the two species ranges between  <dig> , for global, and  <dig>  %, for local alignments.table  <dig> statistics of sequence identity between xla <dig>  and xtr <dig>  genome assemblies.
the sampling size of couples of aligned blocks between x. tropicalis and x. laevis was  <dig>   for all chromosomes
fig.
<dig> boxplot of the global and local alignment sequence identities of the  <dig>  samples of pairs of blocks from all chromosomes.
the box in the boxplot concentrates 50 % of the data.
the whiskers are  <dig> x the length of the box.
the red crosses represent outliers
discussion
in this work we have used x. tropicalis first  <dig> scaffolds  as reference for the coarse-grained mapping of the  <dig> largest x. laevis scaffolds .
using this strategy, we were not only able to map the genes and calculate the conserved synteny of orthologs between these two species but also estimate the percentage of global identity, inversions and repetitions.
taken together, this newly assembled map represents a useful tool for the integration between biochemical, physiological, genetic and genomics data between x. laevis and x. tropicalis.
the map
the expected alignment rate is around  <dig>  considering the rate of genome length between the two species.
our data show a similar alignment rate of  <dig> .
it was also expected the length rate between x. laevis respect to x. tropicalis, i.e., the rate between the length of the scaffolds that align, to be  <dig>  but rather we calculated a length rate of  <dig> .
it is possible that this difference either reflects evolutionary features such as genome rearrangements, translocations, deletions and fusions  <cit> , or are associated with assembly artifacts.
the gaps in xenopus genomes impinge on mapping and gene identification  <cit> .
about  <dig>  % of x. tropicalis and  <dig>  % of x. laevis genomes were used for the mapping.
if we assume that the two genomes are two random sequences of the same size, it is expected that  <dig>  x  <dig>  =  <dig>  % of x. tropicalis genome actually aligns.
the alignment coverages in x. tropicalis and x. laevis genomes is  <dig>  and  <dig>  %, respectively, lower than expected.
the non-aligned blocks, or misalignments, may be due to recombination, deletion or insertion of sequences in both species  <cit> .
whole genome duplication is known to cause recombination and pseudogenization among other adaptive processes  <cit> .
the rearrangements that happened in segments either smaller than 5kbp in one single block or ≥5kbp and ≤10kbp combined in two consecutive blocks might not align with a score over the drop-off score in cgaln.
repetitions and inversions
the meaning of the repetition figures is that  <dig> mbp from x. tropicalis, are aligned with  <dig> mbp in x. laevis, and blocks of 5kbp in that sequences are repeated at least once in additional  <dig> mbp in the x. laevis.
regarding inversions,  <dig> mbp is the estimated length between the two species.
however, this is an underestimate as the inversion identification relies on the colonies aligned, and these only represent a subset of the inversions.
inversions represent 7 % of the aligned portion of x. tropicalis genome and 3 % of the aligned portion of x. laevis genome.
these figures depend on the assembly quality; therefore will probably change in the next releases of xenopus assemblies .
inversions and repetitions are associated with evolutionary rearrangement events  <cit> .
each chromosome alignment , assuming a correct assembly, reveals a few large rearrangements.
in a few cases, for example in chromosome  <dig>  chromosomes l and s show the same general pattern, which suggests that these rearrangements took place before the genome duplication event in the common ancestor between xenopus species.
in other cases, the differences between l and s chromosomes, for example chromosome  <dig>  indicate a rearrangement after the genome duplication event.
the alignments of l and s chromosomes against x. tropicalis chromosomes  <dig> and  <dig> show the fusion point in x. laevis.
the patterns suggest that the chromosomes fusion event took place before the genome duplication event.
often, the border regions of large rearrangements contain long repetitions in the order of  <dig> to 106 bp.
additional analysis of the border regions of these hypothetical rearrangements may confirm them, further validating the assembly.
previous assembly releases
assembly releases xtr <dig>  and xla <dig> , available in  <dig>  were coarse-aligned and analyzed using the same methodology described in this work.
the sequences aligned included the largest  <dig>  from xla <dig>  and the largest  <dig> scaffolds from xtr <dig> , which constitute around 80 % of each genome.
the map had an overall coverage of about 50 % of both genome sequences .
this suggests that new assembly releases may change alignment coverage significantly.
the estimation of inversions was 58 %, largely due to the lack of contiguity of xla <dig>  assembly.
other map features, like alignment rate, repetitions, percentage of sequence identity and gene synteny estimated between the genomes, as expected, confirm the results drawn with releases xtr <dig>  and xla <dig> , used in this work.
additional map validation was performed using fish results from  <cit> .
as the updated versions xla <dig>  and xtr <dig>  were already refined by fluorescence in situ hybridization  experiments  <cit> , such validation was not needed in this study.
CONCLUSIONS
overall, our results indicate that the final map aligns between  <dig> and  <dig>  % of x. tropicalis and x. laevis total genome length despite the fact that the two species are close to be completely sequenced.
the current map allowed an estimation of genome sequence identity between these species ; the location of  <dig>  genes of x. laevis and  <dig>  genes in x. tropicalis, , the automatic annotation of the transcripts of both species, and the calculation of the conserved synteny between the two frog species verifying the correspondent positions of  <dig>  pairs of orthologous genes , making this a useful source for future comparative studies between x. laevis and x. tropicalis.

 african clawed frogs comprise more than twenty species of frogs native to sub-saharan africa. the most studied species in this genus are xenopus laevis and more recently xenopus tropicalis. xenopus species have been an important model in cell biology, development, genetics and genomics. these species are an attractive model in these areas based on the ability to study embryos at all developmental stages, the presence of large eggs in abundant quantities throughout the year and the remarkable regenerative capacity in the tadpole. xenopus research has set key principles in gene regulation and signal transduction, embryonic induction, morphogenesis and patterning as well as cell cycle regulation. historically, x. laevis has been considered one of the main animal models for developmental, cell, electrophysiology and biomedical studies. however, this species presents a challenge for genomics analyses and genetics due to the allotetraploid nature of its genome and its long life cycle. the haploid genome of x. laevis has been sequenced to  % and consists of chromosomes and gbp. current assembly of the x. laevis genome consists in scaffolds in the xenbase release. this release includes the identification of l and s chromosomes from the new nomenclature by matsuda et. al.. the x. laevis transcriptome counts with primary transcript sequences. the annotation of the transcripts, in the current release, include the identification of the genes known to be duplicated, that belong to chromosomes l and s. one limitation of x. laevis, however, has been the lack of systematic genetic studies to complement molecular and cell biology investigations. work with the closely related diploid frog x. tropicalis has attempted to address this limitation. x. tropicalis is a diploid organism with chromosomes and a gbp long haploid genome. currently,  % of the genome has been sequenced, consisting of scaffolds in xenbase release. the first and longest scaffolds correspond to  % of contiguous sequences of the haploid chromosomes in the x. tropicalis genome. this organism has transcript sequences. the easy molecular tractability of genomic features of x. tropicalis has allowed integration of some genetic, biochemical, phenotypic and evolutionary data in these two species. however, correspondence is not always expected between genomic data in x. tropicalis and the duplicated and divergent genome of x. laevis. in the case there is correspondence, establishing it at a genome level is required. this cannot be done without a physical map between both genomes. no comprehensive comparative analyses using genomic sequencing mapping have been conducted for x. laevis and x. tropicalis. aiming at facilitating such analysis, we have set out to build a comparative coarse-grained physical map between these two species. to this end, we aligned the chromosomes from x. laevis assembly xla to the chromosomes from x. tropicalis assembly xtr and estimated percentage of sequence identity, repetitions, inversions and synteny of mapped genes between the two species. finally, we validated the map theoretically through the synteny of maximal unique matches. as a whole, our results convey the suitability of this newly assembled map for comparative studies between these two species, bridging a long-standing gap for the integration of biochemical, genetic and genomics data in xenopus.  in this work we have performed a comparative analysis between the two frog genomes after mapping by a coarse-grain alignment method the chromosome sequences of x. laevis on the chromosome sequences from x. tropicalis and semi automatic annotation of their transcripts to complement the map information. the analyses include a validation of the map, estimations of percentage of sequence identity, repetitions, inversions and synteny between the two genomes.fig. a chart summarizing the workflow from the two xenopus assemblies to the map and the analyses the map as x. laevis genome is around times the length of x. tropicalis genome, is also the expected rate of added lengths of the blocks aligned between the two species. this rate depends on the alignment drop-off score, x, chosen. a resulting rate larger than suggests a loose alignment. on the other hand, a resulting rate smaller than suggests a strict alignment. the drop-off score x =  rendered an average alignment length rate of, which is close to the expected rate. however, the rate between the lengths of the chromosomes from x. laevis respect to x. tropicalis is, larger than expected.table summary of the coarse-grained map between xla chromosomes on xtr chromosomes. the length units are in blocks. each block corresponds to a sequence of length kbp. xtr; xla; chr ain x. laevis, chromosomes and from x. tropicalis become fused and duplicated. they were named chr9_10l and chr9_10s chromosomes in xla. the same set was aligned to x. tropicalis chromosome and chromosome 10 a coarse-grained dotplot alignment between x. laevis scaffolds and each x. tropicalis chromosome scaffold shows graphically part of the information in table . although the alignments seem to be contiguous, overall  % of x. tropicalis chromosomes did not align to x. laevis chromosomes. in supplement to this figure, the proportion of x. tropicalis chromosomes covered by x. laevis was  %. this proportion, combined with the completion of  % of the x. tropicalis genome, results that  % of x. tropicalis whole genome is actually aligned by x. laevis blocks. a similar coverage of  % was obtained for x. laevis chromosomes.fig. dotplot alignments of each xla  l and s chromosomes to each xtr chromosome. a red dot represents a block alignment between x. laevis and x. tropicalis chromosomes. the alignments are not at the same scale conservation between x. tropicalis and x. laevis as the resulting alignment depends on the drop-off value used, we aligned all x. laevis scaffolds against all x. tropicalis chromosomes at increasing drop-off score values. the block positions that appear with no conservation are either not aligned or have a score lower than, in which case cannot be distinguished from chance. the maximum drop-off score at which a pair of blocks can be aligned correlates directly with percentage of sequence identity between aligned sequences. however, as the variance of the percentage of sequence identity per drop-off score value is significant, the percentage of sequence identity cannot be reliably predicted from the drop-off score. in spite of this, the maximum drop-off score at which a pair of blocks is aligned can be used as a measure of conservation. from each chromosome, a histogram of maximum drop-off scores or conservation scores was generated and the coverage of alignment for each drop-off was calculated. the average maximum cgaln drop-off score between the aligning zones of the genomes is.. possibly, the histogram of maximum drop-off scores shows a larger than expected proportion of conserved blocks with score of, as that bin accumulates all blocks with drop-off score or higher. chromosome is the shortest chromosome, and the one that has the lowest average conservation and lowest alignment coverage. in order, from highest to lowest average conservation we have x. tropicalis chromosomes: and. this chromosome conservation order changes to and if the averaging only takes into account the aligning blocks.fig. maximum drop-off scores chart of x. laevis blocks on x. tropicalis. a bar-plot of the maximum drop-off score, x, per block position for chromosome b histogram of drop-off scores. c table with the average maximum drop-off scores per chromosome calculated using all blocks, only the aligning blocks, and the coverage of the aligning blocks on x. tropicalis. the cells in grey show the most conserved chromosomes according each column repetitions and inversions as x. laevis genome is the result of whole genome duplication event, it is expected that x. laevis blocks will align each x. tropicalis block. therefore, a block of nucleotides cannot simply be regarded as a block that happens more than once in a genome. three particular cases have to be taken in account: a block from x. tropicalis that aligns to x. laevis is considered a repeat when it is an additional block to an already-aligned first block at one particular scaffold; it belongs to a third scaffold in addition to two previous aligned scaffolds or; it is a combination of the former two cases. in this map, a total of mbp from x. tropicalis are repeated in mbp in the x. laevis aligned genome. inversions are identified only for colonies, i.e., with at least two consecutive aligning blocks. for colonies, a previous check on the scaffold frame is made, as in cgaln only the best out of the reading frames of each x. laevis scaffold is aligned. an inversion is identified when cgaln takes the plus frame of the x. laevis chromosome and a colony is aligned in reverse respect to the x. tropicalis chromosome. because only colonies in reverse can be identified, the inversions counted are an underestimation of the total number of existing inversions. taking into account this limitation, we estimated at least mbp to be inverted between the two genomes. inversions represent 7 % and 3 % of the aligned portion of x. tropicalis or x. laevis genomes, respectively.table summary of repetitions and inversions in the coarse-grained map between xla chromosomes on xtr chromosomes. columns to are sub estimates of the number of repeated blocks from each genome that align on the other genome. columns to are sub estimates of inversions between the genomes validation of the map in order to validate the map between x. laevis and x. tropicalis, we computed a set of common theoretical probes called maximal unique matches between the two genomes and compared their correlative order in the map. the mums generated were identical between species and 250 nt or longer. the distribution of distances between the corresponding positions in the map for the mums gives a measure of how well the correspondence between the genomes was achieved. the generated list of mums has sequences. from those, were mapped on the ten x. tropicalis chromosomes and were mapped on the x. laevis scaffolds; mums, representing  % of the total, are common and mapped to both species. this number is less than expected as it is lower than the proportion of the x. laevis genome mapped. additionally, or  % of the mapped mums on x. laevis are at a distance of ≤5kbp from the corresponding mum in x. tropicalis. one block, or 5kbp, is the resolution of the map. therefore, we estimate that the correspondence between the two sets of scaffolds was achieved in  % of the map. application of the map: conserved synteny and gene rearrangements to calculate conserved synteny, a set of orthologous genes between two species is required. orthologous genes were found through bidirectional-best-hit using blastn. a subset of these, genes, map on the x. tropicalis chromosomes. out of all x. laevis transcripts, only map on x. tropicalis chromosomes. from these, are orthologous genes and present in at least pairs of consecutive orthologous genes mapped in the same x. laevis chromosome. this set was our orthologous genes sample for synteny estimation. we found that orthologous genes, or  % of the sample, are syntenic between the two species.table distribution of xla transcripts according to its mapping on xtr chromosomes assembly. a transcript is considered partially aligned if only one of the blocks, either the one including the start or the stop position, is aligned. a transcript does not align on x. tropicalis if neither of the blocks that include start or stop positions, is aligned because the intergenic distance is one of the main determinants of order conservation, three distances were measured between pairs of orthologous genes: 1) distance between two consecutive genes in x. laevis; 2) distance between two consecutive genes in x. tropicalis and; 3) distance between x. laevis start block position projected on x. tropicalis and its orthologous gene start block.fig. three distances measured between the consecutive genes in x. laevis, a and b as intergenic-distance, igd; between two consecutive genes in x. tropicalis, a’ and b’ as igd and; the distance between orthologous genes in both genomes d the relative error of the distance between two consecutive genes in x. laevis respect to x. tropicalis was calculated with the first two distances. the mean relative error was  %. this means that regardless the absolute distance between two consecutive orthologous genes in x. tropicalis, the corresponding consecutive genes in x. laevis are, in average, ±  % of that distance apart.  % of the orthologous pairs of genes are in the corresponding block position according to the map. in the case of the distribution for the third measured distance, it was found that orthologous genes are mapped, in average 9kbp, and that 95 % of the orthologous genes are at most 55kbp apart. for comparison, the confidence interval of lengths, at 95 %, of xenopus genes are between and 15kbp. percentage sequence identity between the two species based on the calculated mapping between the two species, and to assess more precisely the sequence conservation, a random sample containing 100mbp of matching blocks were aligned by using the global needleman-wunsch and local smith-waterman dynamic programming algorithms. the aim was to estimate, respectively, upper and lower references of the sequence identity between the two xenopus species. for the two types of alignments, median percentage sequence identities are similar, both per chromosome and in total. the distributions for global and local alignment overlap. the medians are and 43 %, respectively. in average, the percentage sequence identity shared by the two species ranges between, for global, and  %, for local alignments.table statistics of sequence identity between xla and xtr genome assemblies. the sampling size of couples of aligned blocks between x. tropicalis and x. laevis was for all chromosomes fig. boxplot of the global and local alignment sequence identities of the samples of pairs of blocks from all chromosomes. the box in the boxplot concentrates 50 % of the data. the whiskers are x the length of the box. the red crosses represent outliers discussion in this work we have used x. tropicalis first scaffolds as reference for the coarse-grained mapping of the largest x. laevis scaffolds. using this strategy, we were not only able to map the genes and calculate the conserved synteny of orthologs between these two species but also estimate the percentage of global identity, inversions and repetitions. taken together, this newly assembled map represents a useful tool for the integration between biochemical, physiological, genetic and genomics data between x. laevis and x. tropicalis. the map the expected alignment rate is around considering the rate of genome length between the two species. our data show a similar alignment rate of. it was also expected the length rate between x. laevis respect to x. tropicalis, i.e., the rate between the length of the scaffolds that align, to be but rather we calculated a length rate of. it is possible that this difference either reflects evolutionary features such as genome rearrangements, translocations, deletions and fusions, or are associated with assembly artifacts. the gaps in xenopus genomes impinge on mapping and gene identification. about  % of x. tropicalis and  % of x. laevis genomes were used for the mapping. if we assume that the two genomes are two random sequences of the same size, it is expected that x  =   % of x. tropicalis genome actually aligns. the alignment coverages in x. tropicalis and x. laevis genomes is and  %, respectively, lower than expected. the non-aligned blocks, or misalignments, may be due to recombination, deletion or insertion of sequences in both species. whole genome duplication is known to cause recombination and pseudogenization among other adaptive processes. the rearrangements that happened in segments either smaller than 5kbp in one single block or ≥5kbp and ≤10kbp combined in two consecutive blocks might not align with a score over the drop-off score in cgaln. repetitions and inversions the meaning of the repetition figures is that mbp from x. tropicalis, are aligned with mbp in x. laevis, and blocks of 5kbp in that sequences are repeated at least once in additional mbp in the x. laevis. regarding inversions, mbp is the estimated length between the two species. however, this is an underestimate as the inversion identification relies on the colonies aligned, and these only represent a subset of the inversions. inversions represent 7 % of the aligned portion of x. tropicalis genome and 3 % of the aligned portion of x. laevis genome. these figures depend on the assembly quality; therefore will probably change in the next releases of xenopus assemblies. inversions and repetitions are associated with evolutionary rearrangement events. each chromosome alignment, assuming a correct assembly, reveals a few large rearrangements. in a few cases, for example in chromosome chromosomes l and s show the same general pattern, which suggests that these rearrangements took place before the genome duplication event in the common ancestor between xenopus species. in other cases, the differences between l and s chromosomes, for example chromosome indicate a rearrangement after the genome duplication event. the alignments of l and s chromosomes against x. tropicalis chromosomes and show the fusion point in x. laevis. the patterns suggest that the chromosomes fusion event took place before the genome duplication event. often, the border regions of large rearrangements contain long repetitions in the order of to 106 bp. additional analysis of the border regions of these hypothetical rearrangements may confirm them, further validating the assembly. previous assembly releases assembly releases xtr and xla, available in were coarse-aligned and analyzed using the same methodology described in this work. the sequences aligned included the largest from xla and the largest scaffolds from xtr, which constitute around 80 % of each genome. the map had an overall coverage of about 50 % of both genome sequences. this suggests that new assembly releases may change alignment coverage significantly. the estimation of inversions was 58 %, largely due to the lack of contiguity of xla assembly. other map features, like alignment rate, repetitions, percentage of sequence identity and gene synteny estimated between the genomes, as expected, confirm the results drawn with releases xtr and xla, used in this work. additional map validation was performed using fish results from. as the updated versions xla and xtr were already refined by fluorescence in situ hybridization experiments, such validation was not needed in this study.  overall, our results indicate that the final map aligns between and  % of x. tropicalis and x. laevis total genome length despite the fact that the two species are close to be completely sequenced. the current map allowed an estimation of genome sequence identity between these species; the location of genes of x. laevis and genes in x. tropicalis,, the automatic annotation of the transcripts of both species, and the calculation of the conserved synteny between the two frog species verifying the correspondent positions of pairs of orthologous genes, making this a useful source for future comparative studies between x. laevis and x. tropicalis. 
however, for genetic studies, xenopus tropicalis has been the experimental model of choice because it shorter life cycle and due to a more tractable genome that does not result from genome duplication as in the case of x. laevis. taken together, our results make possible to establish the correspondence between and  % of both genomes, percentage of identity, synteny and automatic annotation of transcripts of both species, providing a new and more comprehensive tool for comparative analysis of these two species, by allowing to bridge molecular genetics data among them. there is expectation for a comparative physical map that can be used as a rosetta stone between x. laevis genetic studies and x. tropicalis genomic research. today, although still organized in a large number of scaffolds, nearly 85 % of x. tropicalis and 89 % of x. laevis genomes have been sequenced. in this work, we have mapped using coarse-grained alignment the chromosomes of x. laevis, release, on the reference scaffolds representing the haploid genome of x. tropicalis, release. after validating the mapping with theoretical data, and estimating reference averages of genome sequence identity, to 44 % between the two species, we have carried out a synteny analysis for orthologous genes.
 the clawed african frog xenopus laevis has been one of the main vertebrate models for studies in developmental biology. however, for genetic studies, xenopus tropicalis has been the experimental model of choice because it shorter life cycle and due to a more tractable genome that does not result from genome duplication as in the case of x. laevis. today, although still organized in a large number of scaffolds, nearly 85 % of x. tropicalis and 89 % of x. laevis genomes have been sequenced. there is expectation for a comparative physical map that can be used as a rosetta stone between x. laevis genetic studies and x. tropicalis genomic research.  in this work, we have mapped using coarse-grained alignment the chromosomes of x. laevis, release, on the reference scaffolds representing the haploid genome of x. tropicalis, release. after validating the mapping with theoretical data, and estimating reference averages of genome sequence identity, to 44 % between the two species, we have carried out a synteny analysis for orthologous genes. we found that  % of genes are in the same organization.  taken together, our results make possible to establish the correspondence between and  % of both genomes, percentage of identity, synteny and automatic annotation of transcripts of both species, providing a new and more comprehensive tool for comparative analysis of these two species, by allowing to bridge molecular genetics data among them. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords xenopuslaevistropicalisassemblycoarse-grainedalignmentmapsyntenygenomesequencesfondecyt31304413140005riadi gonzalo ossandón francisco issue-copyright-statement© the author 2016 