BACKGROUND
the speed and affordability of sequencing and improved software, including more efficient genome assemblers, have led to a democratization of genomics, enabling individual research groups to create de novo genome assemblies  <cit> .
the first published de novo assemblies for non-model organisms using pure massively parallel sequencing approaches  appeared in 2010- <dig> and included diverse species such as giant panda  <cit> , turkey  <cit> , woodland strawberry  <cit>  and atlantic cod  <cit> .
numerous genome assemblies from a myriad of non-model plants, invertebrates and vertebrates are now available, including examples of genomes that are difficult to assemble, e.g.
the extremely large genomes of bread wheat  <cit>  and norway spruce  <cit> , the highly heterozygous genome of oyster  <cit>  and the tetraploid and repetitive salmon genome  <cit> .
these genome assemblies have provided exciting new biological findings, including the first example of a vertebrate immune system, that of atlantic cod, which lacks mhc  class ii  <cit> , untangling of the events of multiple hybridizations shaping the ancestral genomes of bread wheat prior to domestication  <cit>  and multiple genomes resolving the avian phylogeny, their radiations and investigation of the genetic basis of complex traits  <cit> .
despite the tremendous impact of the high throughput sequencing generated genomes, many of these assemblies are of varying completeness, depending on the purpose for which they have been obtained  <cit> .
in the examples given above, the sizes of the scaffold sequences are usually far shorter than chromosome arm lengths.
most of these genomes have scaffold n <dig> lengths  in the range of  <dig> kbp– <dig>  mbp, although some avian genomes have n <dig> scaffold lengths up to  <dig> mbp, approaching chromosome arm lengths.
however, contig n <dig> lengths are far shorter and in the range of 3– <dig> kbp.
the presence of repetitive dna is the most important factor contributing to fragmented genome assemblies  <cit> .
assembly algorithms might not resolve repetitive regions if they are longer than the read length, and this in particular affects the assembly of sequencing data from short-read technologies such as the illumina platform .
repetitive regions can be divided into two classes, interspersed and tandem repeats.
interspersed repeats, including transposable elements , occur across the genome and are present in all vertebrate genomes, comprising from  <dig> to 55% of their assemblies  <cit> .
tandem repeats  are sequences with a repeat unit repeated more than two times in tandem.
eukaryotic genomes typically consist of  <dig>  to 3% trs, and trs can be classified into microsatellites, also called simple repeats, or short tandem repeats ; minisatellites  and satellite repeats   <cit> .
trs mutate by adding or removing full repeat units and their mutation rates can be  <dig> to  <dig>  fold higher than for the remainder of the genome  <cit> .
the heterozygosity caused by tr mutations, in addition to other types of heterozygosity, are also likely to have confounding effects on the contiguity of genome assemblies  <cit> .
long-read sequencing technologies such as pacbio and oxford nanopore address the drawbacks of short-read technologies by enabling read-through of larger repeat regions, and are therefore particularly well-suited for de novo genome assembly  <cit> .
combining moderate amounts of pacbio coverage  with other sequencing data can dramatically improve the contiguity of an assembly  <cit> .
more extensive coverage in long reads  has enabled assemblies of vertebrate genomes to approach complete chromosome arms , although the associated costs are substantial.
a sequencing strategy including long-reads is recommended to aid in reducing the fragmentation typical of de novo genome assemblies based on a short-read technology only.
regardless of sequencing strategy, use of a genetic linkage map, or an optical map, can place contigs or scaffolds into chromosome-sized reconstructions, called linkage groups, a prerequisite for large-scale genome comparisons between species  <cit> .
the first release of the atlantic cod  genome was sequenced and assembled solely with the  <dig> sequencing technology  <cit>  and annotated by the ensembl project  <cit>  .
the  <dig> mbp assembly was fragmented, with a contig n <dig> of  <dig>  kbp and 27% of bases in gaps.
the genome assembly contained  <dig> % tes and  <dig> % trs .
an increased abundance of short unit size trs at the contig termini , and at the gaps in scaffolds  indicate that these repeats contributed to the observed level of fragmentation.
a more contiguous reference genome for atlantic cod, preferably with chromosome-level reconstructions, will facilitate re-sequencing efforts addressing population genomics investigations, including the detection of structural variants, introgression and hybridization between species, as well as improve comparative genomic investigations relying on synteny.
moreover, it will also enable an annotation with more complete gene models and allow for a better understanding of the lack of sequence contiguity in gadmor <dig>  to achieve this, we created several assemblies using different combinations of illumina,  <dig> and pacbio sequencing technologies, sanger bac-end sequences, and a suite of assembly programs.
as often is the case , no single assembly outperformed the others in all criteria , thus a reconciled assembly was created to integrate the best characteristics of four draft assemblies.
this new assembly  has a fifty-fold improvement of the contig n <dig> length of gadmor <dig>  and eight times longer scaffold n <dig> and one sixteenth the number of bases in gaps than gadmor <dig>  a linkage map  was used to order and orient the scaffolds into linkage groups.
the new genome assembly and annotation reveal a high content of trs compared to other vertebrates and most notably in promoter regions and amino acid coding sequences.
many of these trs are heterozygous, and we propose this has implications for understanding local adaptation at a population level.
RESULTS
an improved genome assembly for atlantic cod
in addition to already existing sequencing data for the wild-caught individual from the north east-arctic population described in  <cit>  , we added sequencing data from illumina  and pacbio   obtained from dna isolated from the same individual.
different assembly strategies were used: a newbler assembly with  <dig> and sanger bac-end sequences as input , an allpaths-lg  <cit>  assembly with the illumina sequences only , a celera assembler  <cit>  assembly with  <dig> and illumina sequences  and a celera assembler assembly with  <dig> paired reads, illumina reads and raw, uncorrected pacbio reads  .
for each of the individual assemblies, different combinations of the assembly improvement programs pilon  <cit>  and pbjelly  <cit>  were applied to improve the consensus sequence and to close gaps .
the properties of these assemblies were assessed using multiple tools: 1) methods based on the mapping of read datasets to an assembly, frc
bam  <cit>  and reapr  <cit> ; 2) by comparing a transcriptome to an assembly, isoblat   <cit> ; 3) by comparing the assembly to a linkage map ; 4) and determining presence and completeness of conserved eukaryotic and actinopterygii  gene sets, cegma  <cit>  and busco  <cit>  .
based on these evaluations, each assembly had distinct properties, and none is superior for all metrics.
for instance, the newb <dig> assembly has the longest scaffold n <dig> and the lowest number of conflict sequences .
in contrast, the ca454pb outperforms the other assemblies based on contig n <dig>  yet has a lower scaffold n <dig> and higher number of sequences conflicting with the linkage map  .
existing assembly reconciliation tools are limited to combining two assemblies  <cit>  and do not perform satisfactorily.
to obtain the best possible assembly, i.e., to integrate the information recovered by the different assemblies, we developed a novel assembly reconciliation method.
this method involved an all-against-all alignment of the assemblies using mugsy  <cit>  after splitting the different assemblies in locations where they were in conflict with the linkage map  and removing sequences shorter than  <dig> bp.
the resulting alignment graph structure was traversed following the path from one of the original assemblies , yielding the sequence from the assembly with the least gaps , while using the alignments with alpilm and newb <dig> in the graph to close gaps and extend scaffolds.
the scaffold module from sga  <cit>  was applied on the resulting merged assembly using all paired reads , and pilon  <cit>  was used to improve per-base accuracy and to close or reduce gaps.
the resulting assembly was ordered and oriented based on a linkage map of  <dig> snps  placing 93% of the sequences into  <dig> linkage groups .
comparisons of assembly statistics for the final, reconciled assembly  and the original four , show that gadmor <dig> outperforms all other assemblies on all quality metrics apart from scaffold n <dig>  and cegma gene content .
based on an overall assessment of quality, gadmor <dig> combines the best features of each of the four original assemblies without loss of quality .
fig.
<dig> contig and scaffold n <dig> lengths of the different cod assemblies.
gadmor <dig> was created by following the sequences in ca454ilm in a path through a graph created from a multiple alignment of the four original assemblies, and outputting the contig sequences from ca454pb for each alignment.
newb <dig> and alpilm were used to extend the scaffolds, see table 1
frc
bam
d
gadmor1f
gadmor2g
acegma annotates  <dig> highly conserved eukaryotic genes
bbusco annotates  <dig>  actinopterygii specific genes
creapr analyses the discordance between the expected order, orientation and distance of mapped paired reads, with detected potential errors, fewer is better
d
frc
bam uses a similar approach as reapr, with total number of features , fewer is better
enumber of sequences mapping to more than one linkage group or to multiple linkage groups, fewer is better
ffrom  <cit>
g93% of the gadmor <dig> assembly is additionally oriented and ordered into  <dig> linkage groups
the gadmor <dig> assembly has a fifty-fold longer contig n <dig> and eight-fold longer scaffold n <dig> compared to the gadmor <dig> assembly  <cit> .
this has dramatic consequences for the sequence contiguity; for instance, a 100kbp region containing the hoxc cluster is a single contig in gadmor <dig>  while it previously consisted of  <dig> contigs and  <dig> gaps in gadmor <dig> .
fig.
<dig> the hoxc cluster in gadmor <dig> and gadmor <dig>  blocks of dark and light blue are contig sequences, white blocks are gaps and red lines are tandem repeats.
gene models are sketched at the top of the figure.
this region is a single contig in gadmor <dig> and  <dig> contigs in gadmor <dig>  tandem repeats are at the borders between almost all gaps and contigs in gadmor1
genome size
estimation of genome size with odd-sized k-mers from  <dig> to  <dig> with sga preqc  <cit>  on the  <dig> bp insert size,  <dig> bp length, paired end illumina reads , resulted in a genome estimate of  <dig> mbp ± <dig> mbp .
the assembler allpaths-lg estimated the genome to be  <dig> mbp based on the k-mer distribution of the  <dig> bp insert size,  <dig> bp length, paired end illumina reads .
both estimates are lower than previous ones based on feulgen image analysis densitometry at  <dig>  pg or  <dig> mbp  <cit>  and a k-mer analysis based on  <dig> reads, which resulted in a  <dig> mbp estimate  <cit> .
although the assembly size of the gadmor <dig> at ensembl is  <dig> mbp with  <dig> % gaps  <cit> , the amount of sequence in contigs is  <dig> mbp , considerably closer to the sga preqc estimate.
the likely explanation for the large size of gadmor <dig> is that many of the contigs could not be placed into a scaffold, and a gap was created at that locus instead.
these unplaced contigs are included in the output, resulting in loci represented twice in the assembly, once as a gap and once as a contig.
the assemblies created in this study all span approximately  <dig> mbp, which is similar to the allpaths-lg estimation.
<dig> mbp is 71% of the earlier estimation based on feulgen image analysis densitometry, and in line with similar results in platyfish   <cit>  and in northern pike   <cit> .
in addition, cegma  <cit>  and busco  <cit>  find 89-93% of conserved genes , likely reflecting the approximate completeness of the genome assembly.
annotation
we annotated  <dig>  gene models with maker <dig>  <cit> , obtaining a final set of  <dig>  predicted genes after discarding gene models with low support .
compared to gadmor <dig>   <cit> , the gadmor <dig> annotation contains more predicted genes and significantly more sequence in the predicted transcriptome .
the predicted transcripts are substantially longer and without gaps .
a genome browser enabling access to the genome and the annotation is available  <cit> .
asum of bases in transcripts with utrs
bhalf the transcriptome is in sequences of this length or longer, with utrs
cgaps represented as ’n’s in annotated transcripts
dnumber  of conserved actinopterygii genes detected out of a total of  <dig>
ewhen excluding pseudogenes, alternative transcripts, etc., the number of protein-coding genes is  <dig>
fprotein-coding genes only
heterozygosity
illumina paired-end reads with  <dig> bp insert size and  <dig> bp read length were mapped to the gadmor <dig> assembly using bwa-mem  <cit> , and  <dig> , <dig> snps ,  <dig>  mnps ,  <dig>  indels  and  <dig>  complex regions  with quality ≥ <dig> were called using freebayes  <cit> .
with  <dig> , <dig> snps, this corresponds to a  heterozygosity rate of  <dig> ×10− <dig> .
the indel rate in atlantic cod is  <dig>  ×10− <dig> .
sticklebacka
miiuy croakerb
atlantic herringc
ciona savignyi
d
ciona savignyi
e
afrom  <cit>
bfrom  <cit>
cfrom  <cit>
dfrom  <cit>
efrom  <cit> , with haplotype assembly and merging
we also called indels based on pacbio sequencing reads using blasr  <cit>  and pbhoney  <cit> .
<dig>  indels of size ≥ <dig> bp were found, at a rate of  <dig>  ×  <dig> − <dig> indels/base, or one indel ≥ <dig> bp every  <dig>  bp on average.
repeat content
we created a repeat library using a combination of repeatmodeler  <cit> , ltrharvest  <cit> , ltrdigest  <cit>  and transposonpsi  <cit>  and known eukaryotic te sequences from repbase  <cit>  .
this library masked  <dig> % of the genome assembly , with  <dig> % classified as interspersed repeats  and  <dig> % as trs , both classifications higher than for gadmor <dig> , indicating a more complete genome assembly.
total interspersed repeatsb
agroups of elements covering less than 1% of the genome assembly are not shown
bthis is the sum of all annotated interspersed repeats, including the first four rows plus sines
tr content
we investigated to what extent different assemblers and sequencing technologies affected the numbers of annotated trs.
phobos  <cit>  was used to find all trs with a unit size of 1- <dig> bp, at least  <dig> bp long , in the different cod assemblies .
our results shows that assemblies created with the celera assembler have the largest amount of trs .
fig.
<dig> the density of trs and the size of the assembly for different cod assemblies.
the different assemblies  are all similar in size, around  <dig> mbp, with the exception of the much larger gadmor <dig>  while the amount of sequence in contigs in the different assemblies  differs substantially.
the vertical distance between pairs of points for each assembly equals the amount of sequence in gaps
the most prominent class of trs in gadmor <dig> is dinucleotide trs, which make up  <dig> % of all annotated repeats, followed by mononucleotide, trinucleotide and tetranucleotide repeats that comprise only  <dig> %,  <dig> % and  <dig> %, respectively .
the average length of dinucleotide repeats is  <dig>  ± <dig>  bp, at an average  <dig> % identity.
in total, dinucleotide repeats make up  <dig> % of the entire gadmor <dig> assembly.
newb <dig> and alpilm have a significantly lower amount of, and shorter, trs annotated than the two assemblies created with celera assembler, ca454ilm and ca454pb .
fig.
<dig> the number of tandem repeats categorized based on unit size.
only tandem repeats with unit size 1- <dig> bp are shown.
a unit size of one indicates a mononucleotide tandem repeat, two a dinucleotide, three a trinucleotide, repeats etc.
the horizontal axis denotes the unit sizes of the repeat, while the vertical axis shows the count of the particular repeat
an analysis of gadmor <dig> compared to all genomes in ensembl , including the genome of california sea hare , shows that the atlantic cod genome assembly has an approximately three-fold higher density of trs than the genome assemblies of other vertebrates .
fig.
<dig> the density of trs in genome assemblies, promoters and coding regions.
the assemblies shown here are from ensembl release  <dig>  excluding gadmor <dig>  plus a human genome based on pacbio data, the california sea hare aplysia californica and gadmor <dig> .
the panels show the density  of trs in the whole assembly, coding regions and promoter regions, respectively.
the human pacbio assembly is not included in the gene and promoter analysis because it has no annotation, and the opossum is lacking for technical limitations.
the species marked are oc , hs , hs , cf , do , xt , pf , dr , pm , sc , ac  and gm
trs cause fragmentation of non-pacbio based assemblies
to investigate the possible genomic features associated with gaps in aplilm, ca454ilm, ca454pb, newb <dig>  gadmor <dig> and gadmor <dig> assemblies, we mapped the contigs from each assembly to gadmor <dig> and categorized the intersections between the contig termini  and different annotated features such as snps, indels, trs, tes and lack of sequence coverage.
for gadmor <dig>  contig termini overlap most prominently with regions lacking read coverage by any sequencing technology, and annotated tes.
the ca454pb shows the same pattern, albeit with a larger fraction of contig termini not overlapping any annotation, suggesting that these contigs end in large repeats not resolved by any assembly.
for the other assemblies, the largest fraction of contig termini overlap with trs at percentages that are significantly higher  than the fraction of the gadmor <dig> assembly annotated as such repeats .
as tes might be longer than the read lengths, they may represent a general challenge for most complex genomes .
fig.
<dig> the intersections between contig termini and different annotated features.
the percentage of contig termini  intersecting different annotations of the genome
heterozygous trs
we used lobstr  <cit>  to investigate the occurrence of heterozygous trs  in the sequenced cod genome.
lobstr is designed to analyze trs with unit length of 1- <dig> bp , and uses tandem repeats finder   <cit>  to detect them in the genome assembly.
lobstr both annotates the strs and discovers variation in str length.
in the sequenced individual, lobstr annotated  <dig>  strs that passed filtering , of which  <dig>  were heterozygous.
compared to phobos , lobstr annotated almost twice as many strs, and the distributions of the lengths of strs between the two programs differ largely , with lobstr identifying relatively short strs, and phobos annotating relatively long strs.
given that lobstr is based on the alignment of the  <dig> bp illumina reads, lobstr’s ability to detect heterozygous trs is limited to repeats around  <dig> bp in size  <cit> , however, the average length of a tr in cod is  <dig>  bp .
as an alternative to using lobstr for detecting heterozygous trs, we used the intersection between trs annotated by phobos and indels annotated by either freebayes  or pbhoney .
altogether,  <dig>  indels were detected in the  <dig>  strs  as annotated by phobos, about three times as many as annotated by lobstr.
for trs of unit sizes 1- <dig> bp, there are  <dig>  indels in  <dig>  trs .
our results indicate that at least one-fifth of the trs in the sequenced individual are heterozygous.
trs in genes and promoters
we investigated the intersection of trs and coding regions, and found  <dig>  coding regions in  <dig>  genes contained a tr.
of these trs,  <dig>  trs  intersect an indel as annotated by either mapped pacbio or illumina data.
these heterozygous trs within coding regions are found in  <dig>  genes .
in addition, we investigated the  <dig> kbp sequence upstream of annotated genes .
of the  <dig>  trs identified in these promoter regions,  <dig>   have an indel annotated based on the union of pacbio and illumina data.
discussion
an improved genome assembly for atlantic cod
here we present a new and significantly improved version of the atlantic cod genome assembly with successful integration of data from different sequencing technologies.
the final assembly  was created using a novel reconciliation method, aimed at combining the strengths of four separate assemblies into an integrated assembly maximizing desired metrics, i.e.
contig length, scaffold lengths, gene content and accordance with read data .
the individual assemblies used for the reconciliation were based on different combinations of sequencing technologies and assembly programs, and varied widely in the different studied metrics.
importantly, the inclusion of the long pacbio reads spanning many more repeats than the other sequencing technologies, resulted in an assembly  with a contig n <dig> an order of magnitude longer than the other assemblies, contributing directly to the long contig n <dig> of the final assembly.
to our knowledge, the specific approach used in generating ca454pb, where the raw, uncorrected pacbio reads were first trimmed and then assembled without correction, together with illumina and  <dig> data , has not been previously described.
a similar approach was used in generating one assembly for atlantic salmon , but the sequence in that assembly did not contribute to the final assembly.
end-sequenced bac  libraries provide long-range information in the  <dig> kbp range, and such sequences are available for atlantic cod  <cit> .
the insert size distribution of the bac-end library was bi-modal , which is not handled properly in the celera assembler.
we therefore included these data in the newbler assembly  only, which contributed to this assembly having the longest n <dig> scaffold of the original assemblies.
the assembly using a combination of  <dig> and illumina sequencing reads  was the most complete in regards to genes as found by the assembly validation tools cegma and busco.
while the available illumina sequencing read datasets did not exactly match the recommendations for allpaths-lg  <cit> , the resulting assembly  performed better than gadmor <dig> with regards to n <dig> contig and scaffold metrics.
despite its short contigs and scaffolds, this assembly contributed to the assembly reconciliation process, resulting in longer scaffolds.
our results illustrate a dilemma in obtaining high quality genome assemblies: different combinations of datasets and software using algorithms optimized for certain characteristics of the datasets yield assemblies that are of good quality on different combinations of desired quality criteria, but hardly ever on all  <cit> .
assembly reconciliation helps solve this issue  <cit> , however even our integrated assembly does not rank best on every single metric evaluated.
further improvements in sequencing technology and assembly algorithms are necessary to resolve this problem in genome assembly.
due to the fragmented nature of the first version of the atlantic cod genome, gadmor <dig>  gene-models were reconstructed for the annotation using information from the annotated stickleback genome , and by manual curation .
in contrast, the gadmor <dig> gene models were automatically annotated directly on the genome assembly.
this automated annotation did not annotate pseudogenes, in contrast to the manual curated annotation for gadmor <dig>  the difference in annotation might explain why the cegma validation results are slightly lower for the new reference genome, since well-annotated gene models in stickleback would be transferred to gadmor <dig> .
the gadmor <dig> assembly shows fewer indications of potential assembly errors as detected by frc
bam and in comparison to the linkage map, but more according to the reapr program.
this difference is associated with longer contigs and scaffolds in gadmor <dig>  which enabled reapr to estimate more long-range errors.
the predicted transcriptome is larger in gadmor <dig> , although more genes are found with busco in the gadmor <dig> predicted transcriptome.
busco is designed to detect genes that are often short , which means they are more likely put together properly in the gene-model optimized gadmor <dig> assembly, since longer genes are more likely to be fragmented.
causes of fragmentation of cod assemblies
to understand the fragmented nature of gadmor <dig>  we first focused on the rate of heterozygosity, as substantial differences between the homologous chromosomes of diploid organisms can fragment an assembly  <cit> .
we compared the heterozygosity rate of the gadmor <dig> genome assembly  to three other fish with genomes for which such data is available, i.e.
the miiuy croaker  <cit> , three-spined stickleback  <cit>  and atlantic herring  <cit> , and to the sea squirt ciona savignyi  <cit> , a species with extremely high heterozygosity .
the genomes for the fishes have been assembled to high contiguity .
although a direct comparison may be confounded by the differences in population structure , and by a larger uncertainty connected with calling indels correctly than with snp calls  <cit> , there are substantial differences between the different species.
gadmor <dig> had a n <dig> contig length of  <dig>  kbp , substantially shorter than even ciona savignyi which has an order of magnitude higher snp rate than atlantic cod.
while species with higher snp rates seem to have shorter n <dig> contig length , the sequencing and assembly strategies for the different organisms vary.
for gadmor <dig>  the high snp rate may have had some impact on the fragmentation , but it is not the main explanation.
different combinations of sequencing technology and assemblers vary in their proportion of trs present in the resulting genome assembly .
assemblies with higher density in trs also have more sequence in contigs , indicating that trs are more completely assembled.
the more fragmented assemblies  have a lower density of trs and shorter trs on average, suggesting that trs led to fragmentation of the assembly .
indeed, these assemblies have a much higher proportion  of contig termini intersecting trs  than the tr density of  <dig> % in gadmor <dig> .
only ca454pb and  gadmor <dig> have about 10% of their contig termini intersecting trs.
the remaining gaps in ca454pb and gadmor <dig> are associated with a lack of sequence coverage and tes longer than the pacbio read lengths .
this illustrates the importance of the availability of the pacbio reads, which was the only read type able to span the multitude of trs in the genome.
as illustrated in fig.
<dig>  gadmor <dig> has a much higher contiguity, while a large fraction of gaps in gadmor <dig> are flanked with trs.
thus, our approach to assemble the genome has addressed the fragmentation affecting the gadmor <dig> assembly.
in conclusion, the high occurrence of trs in the cod genome has caused the fragmentation of gadmor <dig> and all assemblies except ca454pb and  gadmor <dig>  without the inclusion of reads obtained from the pacbio technology, or similar sequencing technologies that can span long trs, assembly of genomes with a high density of trs, such as the atlantic cod, to a high sequence contiguity will be significantly more challenging.
the atlantic cod genome reveals an extraordinary high density of trs
we have confirmed and extended previous results showing high genomic densities of strs in atlantic cod  <cit>  by comparison with  <dig> eukaryote genomes .
while most of the species studied have fewer than  <dig> % of bases in trs, california sea hare, kangaroo rat and atlantic cod have more than 6% bases in trs.
atlantic cod has by far the highest density  and frequency  of trs in the whole genome assembly, coding regions and promoters, with only california sea hare having a higher frequency  of trs in promoter regions.
potential role of trs in evolutionary processes in atlantic cod
the mutation rates of trs, and especially strs, are orders of magnitude higher than those of other genomic sequences  <cit> .
in the sequenced individual, we find that one fifth of the annotated trs are heterozygous, with somewhat lower proportions in promoters  and coding regions .
these results are based on the mapping of illumina and pacbio reads, but are likely underestimates.
most of the trs in cod have a short repeat unit that mutate by adding or removing at least one repeat unit, for instance, two nucleotides in the case of dinucleotide repeats.
small differences between two long alleles of a tr would likely not be captured by our analyses, because the illumina reads would not map well to these  <cit> , and the pacbio reads might not give sufficient resolution.
in humans, trs are best known in connection with diseases such as huntington’s disease  <cit> .
in other species, variability  in trs in promoter regions has been associated with diverse phenomena such as behavior in voles  <cit>  and to skull form in dogs  <cit> .
in both saccharomyces cerevisiae and humans, some promoter regions contain trs  <cit> , for which variation in length has been linked to variation in expression  <cit> .
trs in promoter regions may also contribute to expression divergence in great apes  <cit>  and speciation in primates  <cit> .
there is also variability in trs in genes leading to functional variation such as in saccharomyces cerevisiae, where trs in cell-wall genes underlie variation that causes alterations in phenotype, with different genotypes have differences in adhesion, flocculation or biofilm formation  <cit> .
further, in hawaiian mints, variation in a gene coding for a flowering time protein is associated with colonization and radiation of the plant, with longer versions of the gene existing in younger populations and this is suggested to contribute to morphological change and speciation  <cit> .
interestingly, atlantic cod has a higher frequency of trs than these species within both promoters and coding regions .
the sequenced individual was from the north-east arctic cod population, the largest cod population in the world  <cit> , with a large effective population size  <cit> .
extrapolating the high mutation rate of trs, and the observed level of polymorphism in this single individual, suggests that most trs are polymorphic at a population level.
these polymorphic trs contribute substantially to standing levels of genomic variation in atlantic cod populations within and in the vicinity of genes.
CONCLUSIONS
atlantic cod has an extraordinary amount of trs compared to other species.
this repeat content has previously led to complications for assembling the genome.
here, this has been addressed with the usage of the long pacbio sequencing reads and reconciliation of multiple assemblies.
the large amount of trs is likely to have profound evolutionary impact.
in particular, the trs in coding and regulatory regions will drive genetic variation affecting the function or regulation of genes in atlantic cod populations.
it remains to be investigated how cod populations evolve under variable environmental conditions with respect to trs, and whether selection for repeat variation can lead to rapid evolutionary adaptations.

 the speed and affordability of sequencing and improved software, including more efficient genome assemblers, have led to a democratization of genomics, enabling individual research groups to create de novo genome assemblies. the first published de novo assemblies for non-model organisms using pure massively parallel sequencing approaches appeared in 2010- and included diverse species such as giant panda, turkey, woodland strawberry and atlantic cod. numerous genome assemblies from a myriad of non-model plants, invertebrates and vertebrates are now available, including examples of genomes that are difficult to assemble, e.g. the extremely large genomes of bread wheat and norway spruce, the highly heterozygous genome of oyster and the tetraploid and repetitive salmon genome. these genome assemblies have provided exciting new biological findings, including the first example of a vertebrate immune system, that of atlantic cod, which lacks mhc class ii, untangling of the events of multiple hybridizations shaping the ancestral genomes of bread wheat prior to domestication and multiple genomes resolving the avian phylogeny, their radiations and investigation of the genetic basis of complex traits. despite the tremendous impact of the high throughput sequencing generated genomes, many of these assemblies are of varying completeness, depending on the purpose for which they have been obtained. in the examples given above, the sizes of the scaffold sequences are usually far shorter than chromosome arm lengths. most of these genomes have scaffold n lengths in the range of kbp– mbp, although some avian genomes have n scaffold lengths up to mbp, approaching chromosome arm lengths. however, contig n lengths are far shorter and in the range of 3– kbp. the presence of repetitive dna is the most important factor contributing to fragmented genome assemblies. assembly algorithms might not resolve repetitive regions if they are longer than the read length, and this in particular affects the assembly of sequencing data from short-read technologies such as the illumina platform. repetitive regions can be divided into two classes, interspersed and tandem repeats. interspersed repeats, including transposable elements, occur across the genome and are present in all vertebrate genomes, comprising from to 55% of their assemblies. tandem repeats are sequences with a repeat unit repeated more than two times in tandem. eukaryotic genomes typically consist of to 3% trs, and trs can be classified into microsatellites, also called simple repeats, or short tandem repeats; minisatellites and satellite repeats. trs mutate by adding or removing full repeat units and their mutation rates can be to fold higher than for the remainder of the genome. the heterozygosity caused by tr mutations, in addition to other types of heterozygosity, are also likely to have confounding effects on the contiguity of genome assemblies. long-read sequencing technologies such as pacbio and oxford nanopore address the drawbacks of short-read technologies by enabling read-through of larger repeat regions, and are therefore particularly well-suited for de novo genome assembly. combining moderate amounts of pacbio coverage with other sequencing data can dramatically improve the contiguity of an assembly. more extensive coverage in long reads has enabled assemblies of vertebrate genomes to approach complete chromosome arms, although the associated costs are substantial. a sequencing strategy including long-reads is recommended to aid in reducing the fragmentation typical of de novo genome assemblies based on a short-read technology only. regardless of sequencing strategy, use of a genetic linkage map, or an optical map, can place contigs or scaffolds into chromosome-sized reconstructions, called linkage groups, a prerequisite for large-scale genome comparisons between species. the first release of the atlantic cod genome was sequenced and assembled solely with the sequencing technology and annotated by the ensembl project. the mbp assembly was fragmented, with a contig n of kbp and 27% of bases in gaps. the genome assembly contained % tes and % trs. an increased abundance of short unit size trs at the contig termini, and at the gaps in scaffolds indicate that these repeats contributed to the observed level of fragmentation. a more contiguous reference genome for atlantic cod, preferably with chromosome-level reconstructions, will facilitate re-sequencing efforts addressing population genomics investigations, including the detection of structural variants, introgression and hybridization between species, as well as improve comparative genomic investigations relying on synteny. moreover, it will also enable an annotation with more complete gene models and allow for a better understanding of the lack of sequence contiguity in gadmor to achieve this, we created several assemblies using different combinations of illumina, and pacbio sequencing technologies, sanger bac-end sequences, and a suite of assembly programs. as often is the case, no single assembly outperformed the others in all criteria, thus a reconciled assembly was created to integrate the best characteristics of four draft assemblies. this new assembly has a fifty-fold improvement of the contig n length of gadmor and eight times longer scaffold n and one sixteenth the number of bases in gaps than gadmor a linkage map was used to order and orient the scaffolds into linkage groups. the new genome assembly and annotation reveal a high content of trs compared to other vertebrates and most notably in promoter regions and amino acid coding sequences. many of these trs are heterozygous, and we propose this has implications for understanding local adaptation at a population level.  an improved genome assembly for atlantic cod in addition to already existing sequencing data for the wild-caught individual from the north east-arctic population described in, we added sequencing data from illumina and pacbio obtained from dna isolated from the same individual. different assembly strategies were used: a newbler assembly with and sanger bac-end sequences as input, an allpaths-lg assembly with the illumina sequences only, a celera assembler assembly with and illumina sequences and a celera assembler assembly with paired reads, illumina reads and raw, uncorrected pacbio reads. for each of the individual assemblies, different combinations of the assembly improvement programs pilon and pbjelly were applied to improve the consensus sequence and to close gaps. the properties of these assemblies were assessed using multiple tools: 1) methods based on the mapping of read datasets to an assembly, frc bam and reapr; 2) by comparing a transcriptome to an assembly, isoblat; 3) by comparing the assembly to a linkage map; 4) and determining presence and completeness of conserved eukaryotic and actinopterygii gene sets, cegma and busco. based on these evaluations, each assembly had distinct properties, and none is superior for all metrics. for instance, the newb assembly has the longest scaffold n and the lowest number of conflict sequences. in contrast, the ca454pb outperforms the other assemblies based on contig n yet has a lower scaffold n and higher number of sequences conflicting with the linkage map. existing assembly reconciliation tools are limited to combining two assemblies and do not perform satisfactorily. to obtain the best possible assembly, i.e., to integrate the information recovered by the different assemblies, we developed a novel assembly reconciliation method. this method involved an all-against-all alignment of the assemblies using mugsy after splitting the different assemblies in locations where they were in conflict with the linkage map and removing sequences shorter than bp. the resulting alignment graph structure was traversed following the path from one of the original assemblies, yielding the sequence from the assembly with the least gaps, while using the alignments with alpilm and newb in the graph to close gaps and extend scaffolds. the scaffold module from sga was applied on the resulting merged assembly using all paired reads, and pilon was used to improve per-base accuracy and to close or reduce gaps. the resulting assembly was ordered and oriented based on a linkage map of snps placing 93% of the sequences into linkage groups. comparisons of assembly statistics for the final, reconciled assembly and the original four, show that gadmor outperforms all other assemblies on all quality metrics apart from scaffold n and cegma gene content. based on an overall assessment of quality, gadmor combines the best features of each of the four original assemblies without loss of quality. fig. contig and scaffold n lengths of the different cod assemblies. gadmor was created by following the sequences in ca454ilm in a path through a graph created from a multiple alignment of the four original assemblies, and outputting the contig sequences from ca454pb for each alignment. newb and alpilm were used to extend the scaffolds, see table 1 frc bam d gadmor1f gadmor2g acegma annotates highly conserved eukaryotic genes bbusco annotates actinopterygii specific genes creapr analyses the discordance between the expected order, orientation and distance of mapped paired reads, with detected potential errors, fewer is better d frc bam uses a similar approach as reapr, with total number of features, fewer is better enumber of sequences mapping to more than one linkage group or to multiple linkage groups, fewer is better ffrom g93% of the gadmor assembly is additionally oriented and ordered into linkage groups the gadmor assembly has a fifty-fold longer contig n and eight-fold longer scaffold n compared to the gadmor assembly. this has dramatic consequences for the sequence contiguity; for instance, a 100kbp region containing the hoxc cluster is a single contig in gadmor while it previously consisted of contigs and gaps in gadmor. fig. the hoxc cluster in gadmor and gadmor blocks of dark and light blue are contig sequences, white blocks are gaps and red lines are tandem repeats. gene models are sketched at the top of the figure. this region is a single contig in gadmor and contigs in gadmor tandem repeats are at the borders between almost all gaps and contigs in gadmor1 genome size estimation of genome size with odd-sized k-mers from to with sga preqc on the bp insert size, bp length, paired end illumina reads, resulted in a genome estimate of mbp ± mbp. the assembler allpaths-lg estimated the genome to be mbp based on the k-mer distribution of the bp insert size, bp length, paired end illumina reads. both estimates are lower than previous ones based on feulgen image analysis densitometry at pg or mbp and a k-mer analysis based on reads, which resulted in a mbp estimate. although the assembly size of the gadmor at ensembl is mbp with % gaps, the amount of sequence in contigs is mbp, considerably closer to the sga preqc estimate. the likely explanation for the large size of gadmor is that many of the contigs could not be placed into a scaffold, and a gap was created at that locus instead. these unplaced contigs are included in the output, resulting in loci represented twice in the assembly, once as a gap and once as a contig. the assemblies created in this study all span approximately mbp, which is similar to the allpaths-lg estimation. mbp is 71% of the earlier estimation based on feulgen image analysis densitometry, and in line with similar results in platyfish and in northern pike. in addition, cegma and busco find 89-93% of conserved genes, likely reflecting the approximate completeness of the genome assembly. annotation we annotated gene models with maker, obtaining a final set of predicted genes after discarding gene models with low support. compared to gadmor, the gadmor annotation contains more predicted genes and significantly more sequence in the predicted transcriptome. the predicted transcripts are substantially longer and without gaps. a genome browser enabling access to the genome and the annotation is available. asum of bases in transcripts with utrs bhalf the transcriptome is in sequences of this length or longer, with utrs cgaps represented as ’n’s in annotated transcripts dnumber of conserved actinopterygii genes detected out of a total of ewhen excluding pseudogenes, alternative transcripts, etc., the number of protein-coding genes is fprotein-coding genes only heterozygosity illumina paired-end reads with bp insert size and bp read length were mapped to the gadmor assembly using bwa-mem, and, snps, mnps, indels and complex regions with quality ≥ were called using freebayes. with, snps, this corresponds to a heterozygosity rate of ×10−. the indel rate in atlantic cod is ×10−. sticklebacka miiuy croakerb atlantic herringc ciona savignyi d ciona savignyi e afrom bfrom cfrom dfrom efrom, with haplotype assembly and merging we also called indels based on pacbio sequencing reads using blasr and pbhoney. indels of size ≥ bp were found, at a rate of × − indels/base, or one indel ≥ bp every bp on average. repeat content we created a repeat library using a combination of repeatmodeler, ltrharvest, ltrdigest and transposonpsi and known eukaryotic te sequences from repbase. this library masked % of the genome assembly, with % classified as interspersed repeats and % as trs, both classifications higher than for gadmor, indicating a more complete genome assembly. total interspersed repeatsb agroups of elements covering less than 1% of the genome assembly are not shown bthis is the sum of all annotated interspersed repeats, including the first four rows plus sines tr content we investigated to what extent different assemblers and sequencing technologies affected the numbers of annotated trs. phobos was used to find all trs with a unit size of 1- bp, at least bp long, in the different cod assemblies. our results shows that assemblies created with the celera assembler have the largest amount of trs. fig. the density of trs and the size of the assembly for different cod assemblies. the different assemblies are all similar in size, around mbp, with the exception of the much larger gadmor while the amount of sequence in contigs in the different assemblies differs substantially. the vertical distance between pairs of points for each assembly equals the amount of sequence in gaps the most prominent class of trs in gadmor is dinucleotide trs, which make up % of all annotated repeats, followed by mononucleotide, trinucleotide and tetranucleotide repeats that comprise only %, % and %, respectively. the average length of dinucleotide repeats is ± bp, at an average % identity. in total, dinucleotide repeats make up % of the entire gadmor assembly. newb and alpilm have a significantly lower amount of, and shorter, trs annotated than the two assemblies created with celera assembler, ca454ilm and ca454pb. fig. the number of tandem repeats categorized based on unit size. only tandem repeats with unit size 1- bp are shown. a unit size of one indicates a mononucleotide tandem repeat, two a dinucleotide, three a trinucleotide, repeats etc. the horizontal axis denotes the unit sizes of the repeat, while the vertical axis shows the count of the particular repeat an analysis of gadmor compared to all genomes in ensembl, including the genome of california sea hare, shows that the atlantic cod genome assembly has an approximately three-fold higher density of trs than the genome assemblies of other vertebrates. fig. the density of trs in genome assemblies, promoters and coding regions. the assemblies shown here are from ensembl release excluding gadmor plus a human genome based on pacbio data, the california sea hare aplysia californica and gadmor. the panels show the density of trs in the whole assembly, coding regions and promoter regions, respectively. the human pacbio assembly is not included in the gene and promoter analysis because it has no annotation, and the opossum is lacking for technical limitations. the species marked are oc, hs, hs, cf, do, xt, pf, dr, pm, sc, ac and gm trs cause fragmentation of non-pacbio based assemblies to investigate the possible genomic features associated with gaps in aplilm, ca454ilm, ca454pb, newb gadmor and gadmor assemblies, we mapped the contigs from each assembly to gadmor and categorized the intersections between the contig termini and different annotated features such as snps, indels, trs, tes and lack of sequence coverage. for gadmor contig termini overlap most prominently with regions lacking read coverage by any sequencing technology, and annotated tes. the ca454pb shows the same pattern, albeit with a larger fraction of contig termini not overlapping any annotation, suggesting that these contigs end in large repeats not resolved by any assembly. for the other assemblies, the largest fraction of contig termini overlap with trs at percentages that are significantly higher than the fraction of the gadmor assembly annotated as such repeats. as tes might be longer than the read lengths, they may represent a general challenge for most complex genomes. fig. the intersections between contig termini and different annotated features. the percentage of contig termini intersecting different annotations of the genome heterozygous trs we used lobstr to investigate the occurrence of heterozygous trs in the sequenced cod genome. lobstr is designed to analyze trs with unit length of 1- bp, and uses tandem repeats finder to detect them in the genome assembly. lobstr both annotates the strs and discovers variation in str length. in the sequenced individual, lobstr annotated strs that passed filtering, of which were heterozygous. compared to phobos, lobstr annotated almost twice as many strs, and the distributions of the lengths of strs between the two programs differ largely, with lobstr identifying relatively short strs, and phobos annotating relatively long strs. given that lobstr is based on the alignment of the bp illumina reads, lobstr’s ability to detect heterozygous trs is limited to repeats around bp in size, however, the average length of a tr in cod is bp. as an alternative to using lobstr for detecting heterozygous trs, we used the intersection between trs annotated by phobos and indels annotated by either freebayes or pbhoney. altogether, indels were detected in the strs as annotated by phobos, about three times as many as annotated by lobstr. for trs of unit sizes 1- bp, there are indels in trs. our results indicate that at least one-fifth of the trs in the sequenced individual are heterozygous. trs in genes and promoters we investigated the intersection of trs and coding regions, and found coding regions in genes contained a tr. of these trs, trs intersect an indel as annotated by either mapped pacbio or illumina data. these heterozygous trs within coding regions are found in genes. in addition, we investigated the kbp sequence upstream of annotated genes. of the trs identified in these promoter regions, have an indel annotated based on the union of pacbio and illumina data. discussion an improved genome assembly for atlantic cod here we present a new and significantly improved version of the atlantic cod genome assembly with successful integration of data from different sequencing technologies. the final assembly was created using a novel reconciliation method, aimed at combining the strengths of four separate assemblies into an integrated assembly maximizing desired metrics, i.e. contig length, scaffold lengths, gene content and accordance with read data. the individual assemblies used for the reconciliation were based on different combinations of sequencing technologies and assembly programs, and varied widely in the different studied metrics. importantly, the inclusion of the long pacbio reads spanning many more repeats than the other sequencing technologies, resulted in an assembly with a contig n an order of magnitude longer than the other assemblies, contributing directly to the long contig n of the final assembly. to our knowledge, the specific approach used in generating ca454pb, where the raw, uncorrected pacbio reads were first trimmed and then assembled without correction, together with illumina and data, has not been previously described. a similar approach was used in generating one assembly for atlantic salmon, but the sequence in that assembly did not contribute to the final assembly. end-sequenced bac libraries provide long-range information in the kbp range, and such sequences are available for atlantic cod. the insert size distribution of the bac-end library was bi-modal, which is not handled properly in the celera assembler. we therefore included these data in the newbler assembly only, which contributed to this assembly having the longest n scaffold of the original assemblies. the assembly using a combination of and illumina sequencing reads was the most complete in regards to genes as found by the assembly validation tools cegma and busco. while the available illumina sequencing read datasets did not exactly match the recommendations for allpaths-lg, the resulting assembly performed better than gadmor with regards to n contig and scaffold metrics. despite its short contigs and scaffolds, this assembly contributed to the assembly reconciliation process, resulting in longer scaffolds. our results illustrate a dilemma in obtaining high quality genome assemblies: different combinations of datasets and software using algorithms optimized for certain characteristics of the datasets yield assemblies that are of good quality on different combinations of desired quality criteria, but hardly ever on all. assembly reconciliation helps solve this issue, however even our integrated assembly does not rank best on every single metric evaluated. further improvements in sequencing technology and assembly algorithms are necessary to resolve this problem in genome assembly. due to the fragmented nature of the first version of the atlantic cod genome, gadmor gene-models were reconstructed for the annotation using information from the annotated stickleback genome, and by manual curation. in contrast, the gadmor gene models were automatically annotated directly on the genome assembly. this automated annotation did not annotate pseudogenes, in contrast to the manual curated annotation for gadmor the difference in annotation might explain why the cegma validation results are slightly lower for the new reference genome, since well-annotated gene models in stickleback would be transferred to gadmor. the gadmor assembly shows fewer indications of potential assembly errors as detected by frc bam and in comparison to the linkage map, but more according to the reapr program. this difference is associated with longer contigs and scaffolds in gadmor which enabled reapr to estimate more long-range errors. the predicted transcriptome is larger in gadmor, although more genes are found with busco in the gadmor predicted transcriptome. busco is designed to detect genes that are often short, which means they are more likely put together properly in the gene-model optimized gadmor assembly, since longer genes are more likely to be fragmented. causes of fragmentation of cod assemblies to understand the fragmented nature of gadmor we first focused on the rate of heterozygosity, as substantial differences between the homologous chromosomes of diploid organisms can fragment an assembly. we compared the heterozygosity rate of the gadmor genome assembly to three other fish with genomes for which such data is available, i.e. the miiuy croaker, three-spined stickleback and atlantic herring, and to the sea squirt ciona savignyi, a species with extremely high heterozygosity. the genomes for the fishes have been assembled to high contiguity. although a direct comparison may be confounded by the differences in population structure, and by a larger uncertainty connected with calling indels correctly than with snp calls, there are substantial differences between the different species. gadmor had a n contig length of kbp, substantially shorter than even ciona savignyi which has an order of magnitude higher snp rate than atlantic cod. while species with higher snp rates seem to have shorter n contig length, the sequencing and assembly strategies for the different organisms vary. for gadmor the high snp rate may have had some impact on the fragmentation, but it is not the main explanation. different combinations of sequencing technology and assemblers vary in their proportion of trs present in the resulting genome assembly. assemblies with higher density in trs also have more sequence in contigs, indicating that trs are more completely assembled. the more fragmented assemblies have a lower density of trs and shorter trs on average, suggesting that trs led to fragmentation of the assembly. indeed, these assemblies have a much higher proportion of contig termini intersecting trs than the tr density of % in gadmor. only ca454pb and gadmor have about 10% of their contig termini intersecting trs. the remaining gaps in ca454pb and gadmor are associated with a lack of sequence coverage and tes longer than the pacbio read lengths. this illustrates the importance of the availability of the pacbio reads, which was the only read type able to span the multitude of trs in the genome. as illustrated in fig. gadmor has a much higher contiguity, while a large fraction of gaps in gadmor are flanked with trs. thus, our approach to assemble the genome has addressed the fragmentation affecting the gadmor assembly. in conclusion, the high occurrence of trs in the cod genome has caused the fragmentation of gadmor and all assemblies except ca454pb and gadmor without the inclusion of reads obtained from the pacbio technology, or similar sequencing technologies that can span long trs, assembly of genomes with a high density of trs, such as the atlantic cod, to a high sequence contiguity will be significantly more challenging. the atlantic cod genome reveals an extraordinary high density of trs we have confirmed and extended previous results showing high genomic densities of strs in atlantic cod by comparison with eukaryote genomes. while most of the species studied have fewer than % of bases in trs, california sea hare, kangaroo rat and atlantic cod have more than 6% bases in trs. atlantic cod has by far the highest density and frequency of trs in the whole genome assembly, coding regions and promoters, with only california sea hare having a higher frequency of trs in promoter regions. potential role of trs in evolutionary processes in atlantic cod the mutation rates of trs, and especially strs, are orders of magnitude higher than those of other genomic sequences. in the sequenced individual, we find that one fifth of the annotated trs are heterozygous, with somewhat lower proportions in promoters and coding regions. these results are based on the mapping of illumina and pacbio reads, but are likely underestimates. most of the trs in cod have a short repeat unit that mutate by adding or removing at least one repeat unit, for instance, two nucleotides in the case of dinucleotide repeats. small differences between two long alleles of a tr would likely not be captured by our analyses, because the illumina reads would not map well to these, and the pacbio reads might not give sufficient resolution. in humans, trs are best known in connection with diseases such as huntington’s disease. in other species, variability in trs in promoter regions has been associated with diverse phenomena such as behavior in voles and to skull form in dogs. in both saccharomyces cerevisiae and humans, some promoter regions contain trs, for which variation in length has been linked to variation in expression. trs in promoter regions may also contribute to expression divergence in great apes and speciation in primates. there is also variability in trs in genes leading to functional variation such as in saccharomyces cerevisiae, where trs in cell-wall genes underlie variation that causes alterations in phenotype, with different genotypes have differences in adhesion, flocculation or biofilm formation. further, in hawaiian mints, variation in a gene coding for a flowering time protein is associated with colonization and radiation of the plant, with longer versions of the gene existing in younger populations and this is suggested to contribute to morphological change and speciation. interestingly, atlantic cod has a higher frequency of trs than these species within both promoters and coding regions. the sequenced individual was from the north-east arctic cod population, the largest cod population in the world, with a large effective population size. extrapolating the high mutation rate of trs, and the observed level of polymorphism in this single individual, suggests that most trs are polymorphic at a population level. these polymorphic trs contribute substantially to standing levels of genomic variation in atlantic cod populations within and in the vicinity of genes.  atlantic cod has an extraordinary amount of trs compared to other species. this repeat content has previously led to complications for assembling the genome. here, this has been addressed with the usage of the long pacbio sequencing reads and reconciliation of multiple assemblies. the large amount of trs is likely to have profound evolutionary impact. in particular, the trs in coding and regulatory regions will drive genetic variation affecting the function or regulation of genes in atlantic cod populations. it remains to be investigated how cod populations evolve under variable environmental conditions with respect to trs, and whether selection for repeat variation can lead to rapid evolutionary adaptations. 
the first atlantic cod genome assembly published in was one of the early genome assemblies exclusively based on high-throughput pyrosequencing. by combining data from illumina, and the longer pacbio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the atlantic cod genome assembly. indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these trs. the inclusion of pacbio reads combined with the use of multiple assembly programs drastically improved the atlantic cod genome assembly by successfully resolving long trs. the high frequency of heterozygous trs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in atlantic cod populations, which is likely of evolutionary importance. the sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. we show that 21% of the trs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual. since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. compared to other vertebrates, the assembly contains an unusual high density of tandem repeats.
 the first atlantic cod genome assembly published in was one of the early genome assemblies exclusively based on high-throughput pyrosequencing. since then, rapid advances in sequencing technologies have led to a multitude of assemblies generated for complex genomes, although many of these are of a fragmented nature with a significant fraction of bases in gaps. the development of long-read sequencing and improved software now enable the generation of more contiguous genome assemblies.  by combining data from illumina, and the longer pacbio sequencing technologies, as well as integrating the results of multiple assembly programs, we have created a substantially improved version of the atlantic cod genome assembly. the sequence contiguity of this assembly is increased fifty-fold and the proportion of gap-bases has been reduced fifteen-fold. compared to other vertebrates, the assembly contains an unusual high density of tandem repeats. indeed, retrospective analyses reveal that gaps in the first genome assembly were largely associated with these trs. we show that 21% of the trs across the assembly, 19% in the promoter regions and 12% in the coding sequences are heterozygous in the sequenced individual.  the inclusion of pacbio reads combined with the use of multiple assembly programs drastically improved the atlantic cod genome assembly by successfully resolving long trs. the high frequency of heterozygous trs within or in the vicinity of genes in the genome indicate a considerable standing genomic variation in atlantic cod populations, which is likely of evolutionary importance. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. keywords assembly algorithmsassembly consolidationdinucleotide repeatsgadus morhuaheterozygosityindel polymorphismlong-read sequencing technologymicrosatellitespacbiorepetitive dnanorwegian research council199806issue-copyright-statement© the author 2017 