BACKGROUND
next generation sequencing  has spurred a revolution in the development of genomic tools for non-model organisms  <cit> .
in particular, sequencing complete transcriptomes  <cit>  or complexity-reduced fractions of genomes  <cit>  has enabled the identification of genome-wide molecular markers such as single nucleotide polymorphisms  and microsatellites .
such investigations have also addressed fundamental questions in molecular ecology and evolution, such as the genomic basis for speciation  <cit> , morphological variation  <cit> , disease resistance  <cit>  and selection on life history traits  <cit> .
a complete genome sequence is the ultimate genomic tool for a species.
if such a sequence is available it is possible to conduct large-scale, in-depth studies of many important molecular biology processes such as gene expression, transcription regulation, alternative splicing, epigenetic modifications and gene-protein interactions  which are important in ecological studies.
however, applying ngs technologies such as de-novo sequencing on a large eukaryotic genome is still rare, as it represents a considerable investment.
the sheer volume of data generated and the computational facilities needed to assemble and analyse it may limit the number of non-specialized labs that are currently able to embark on such a project.
however, more whole genome studies are needed to address fundamental questions on the evolution of genome organisation, such as which regions are conserved and which regions change when taxa diverge and become separate species.
published whole ngs genomes of non-model organisms include giant panda  <cit> , cod  <cit> , naked mole rat  <cit> , macaque  <cit> , tasmanian devil  <cit> , budgerigar  <cit> , puerto rican parrot  <cit> , heliconius butterfly  <cit> , aye-aye  <cit> , collared flycatcher  <cit> , as well as the  <dig> mammalian genomes recently sequenced at the broad institute  <cit> .
the large number of publically available whole genome sequences from both model and non-model organisms can be used to aid genomic investigations in related organisms.
one approach is to directly transfer the genomic resources from a model organism to the study species, which would then be called ‘genome enabled taxa’  <cit> .
this strategy has been used successfully to develop resources such as microsatellite markers  <cit> , snps  <cit> , microarrays  <cit>  and exon capture arrays  <cit> .
alternatively, the genome sequence from a related model organism can be used in the assembly of short read data from the focal species, a process known as reference guided  assembly  <cit> .
here, we describe a reference guided, whole genome assembly of the black grouse .
we take advantage of the close relationship between this species and the well characterized chicken   and develop a reference guided assembly pipeline to construct a draft genomic sequence.
the black grouse is well studied as a model for ecology and conservation biology  but, until recently, genomic resources for this species have been largely lacking.
this work completes our genomic tools development, previously initiated by characterizing the transcriptome  <cit>  and sequencing the major histocompatibility complex  region  <cit>  in this species.figure  <dig>
male black grouse displaying at a lek.
in theory there are two different ways of using the reference sequence to guide the assembly process.
under an “align-then-assemble” strategy, the reads are first mapped to the reference sequence and clusters of reads mapping to the same location are then extracted and assembled de-novo.
alternatively, in the “assemble-then-align” strategy, the reads are first de-novo assembled and the resulting contigs are then aligned to the reference genome to close gaps and create scaffolds  <cit> .
in our reference guided assembly pipeline  we use a combination of these two approaches by mapping both mate-paired reads and de-novo assembled contigs to the reference genome and combining these alignments to produce the final scaffolds.
this approach has some similarities to the previously published “reference-assisted chromosome assembly”  <cit> .
importantly, we also demonstrate the utility of the solid sequencing technology  for whole genome de-novo sequencing.
due to the short reads produced by this method compared to more widely used  <dig>  and hiseq  sequencers, the solid platform has not been used before for sequencing of vertebrate sized genomes in a non-model organism.
bacterial  <cit>  and fungal  <cit>  genomes have, however, previously been sequenced solely based on this technology.
even though the de-novo assembly of our short read data was fragmented  we were still able to successfully cover a large proportion of the genome using our reference guided approach.
we used the draft genome both to identify a vast number of snps and to perform comparative genomic analyses.figure  <dig>
flow chart of our reference guided genome assembly pipeline.
all reads were first de-novo assembled.
in the second step both long contigs and original mate-paired reads were mapped to the chicken reference genome and merged to produce the black grouse backbone scaffolds.
finally gap-filling was performed by mapping all contigs  back to the backbone scaffolds, producing the draft genome assembly.
in addition all contigs  not mapping to the backbone scaffolds were added to the assembly after removing sequences arising from possible contamination using a blast approach.
for more details about the procedures please see the methods section.
in the figures to the right the chicken reference genome is indicated by the blue line while reads, contigs and scaffolds from the black grouse are shown in red.
the light red parts of the final scaffold indicate regions with gaps  in the black grouse sequence.
within brackets in the boxes to the left are the software used for each different stage of the assembly process.
RESULTS
solid sequencing
the raw sequencing data was comprised of 793 m reads with a read length of 75 bp which were generated for the single-end library, 1642 m reads with read length of 60 bp × 60 bp which were generated for the 2 kb mate-paired library, and 1548 m reads with read length of 60 bp × 60 bp which were generated for the 5 kb mate-paired library.
the raw reads are deposited in the ncbi sequence read archive  under the accession number sra <dig>  after quality and length filtering, 423 m reads  were retained for the single-end library, 320 m  of which were 75 bp in length.
for the 2 kb mate-paired library, 857 m reads  were retained after filtering, and 663 m  of them were 60 bp in length.
for those filtered reads, 519 m  were properly paired, and the rest were only retained as unpaired reads.
for the 5 kb mate-paired library, 847 m reads  were retained after filtering, 648 m  of which were of 60 bp in length.
for those filtered reads, 520 m  were properly paired, and the rest were only retained as unpaired reads.
therefore, 2127 m high quality sequencing reads with the total length of approximately 133 gb were kept in downstream analysis.
if we assume that the genome size of black grouse is similar to that of chicken , the estimated mean sequencing coverage of the black grouse genome was 127x.
reference guided assembly
the reference guided assembly is comprised of several steps, including de novo assembly, reference mapping and the merging of these results .
in the first step, all the 2127 m filtered high quality reads were de novo assembled by soapdenovo.
we were able to generate  <dig> preliminary contigs with a total length of 937 mb.
as expected, the de novo assembly was more fragmented compared to some other studies which also used short-read sequencing technologies  <cit> , this is because in this study we only had three sequencing libraries with a maximum insert size of 5 kbp and the sequencing reads produced by the solid technology were relatively short.
the solid platform is believed to produce high quality reads  <cit> .
all the filtered data we used in our analyses had an error rate not larger than  <dig> %.
however, the short read length seriously affects its performance in pure de novo assembly.
longer sequencing reads produced by platforms such as  <dig>  ion-torrent or pacbio usually produce larger contigs and such data could be used to improve our assembly in the future.
in the next step, we aligned all long contigs to the chicken genome  and were able to map  <dig> of them.
the total mapped length was 438 mb.
at the same time, we also aligned the filtered and properly paired reads from the mate-paired libraries to the chicken genome resulting in 451 m successfully mapped reads.
these two sets of mapped reads were merged and this resulted in a 805 mb black grouse genome backbone scaffold.
finally, we mapped the de novo assembled contigs back to the black grouse backbone scaffolds and had  <dig> of them mapped.
therefore, we succeeded to cover 833 mb  of the 1046 mb chicken genome, and  <dig> of the  <dig> chicken scaffolds .
we covered 826 mb  of the 1001 mb main chicken chromosomes .
in addition, we also retained  <dig> unmapped contigs  with a total length of  <dig>  mb.
the resulting black grouse draft genome assembly consisted of  <dig> scaffolds with a total length of 1022 mb .
the genome assembly is deposited in the ncbi whole genome shotgun  database under the submission number jdsl <dig>  among the scaffolds, the  <dig> largest, corresponding to the chicken chromosomes 1 ~  <dig> and chromosome z, had a total length of 1001 mb .
the average coverage  of the  <dig> chromosomes was  <dig> %.
however, this coverage was not distributed evenly across the chromosome scaffolds or across the chromosomal regions .
we noticed that chromosome  <dig>  chromosome  <dig>  chromosome  <dig> and chromosome z were not well covered.
chromosome z is the avian sex chromosome and chromosome  <dig> harbours the mhc genes  <cit> .
these might be more divergent between black grouse and chicken than the rest of the genome, which may have led to the poor assembly.
for chromosome  <dig>  an additional reason might be that the chicken assembly of this chromosome is still not perfect and contain large n chunks.
furthermore, we have previously shown that when comparing this region among different galliform species, there are several gene copy divergences as well as genomic inversions in the mhc region on chromosome  <dig>  <cit> .
we further examined the quality of the black grouse draft genome, and found that although the sequence coverage was high, the scaffold sequences were highly fragmented .
if the draft genome is split at all ‘n’ sites present on the scaffolds, it has  <dig> continuous sequenced blocks.
this is not unexpected since the solid data was short and we used a reference guided approach to produce the draft genome, and the existing bioinformatic tools available are not mature enough in dealing with this strategy.
the samtools pipeline we used generates consensus sequences solely based on the coordinates of the reference genome, which might introduce a number of additional ‘n’s in the resulted sequences.
in addition, a number of the long ‘n’ chunks are also present in the reference chicken genome, and might thereby be introduced into the black grouse draft genome through the reference guided assembly process.figure  <dig>
heatmap showing the proportion of the regions sequenced on the chromosome scaffolds.
distributions of the continuous sequence-blocks and the interleaved ‘n’ blocks.
the blue lines indicate the total length  of the respective blocks.
the sequenced blocks larger than 10000 bp and the ‘n’ blocks larger than 30000 bp were binned at the end of the axis.
annotation
the fragmented state of the draft genome limited our ability to systematically perform ab-initio predictions of genes or genomic repeats.
instead we used comparative methods to identify the gene regions and the genomic repetitive regions.
from the reciprocal blast result, we found that  <dig> chicken genes had homologs on the black grouse genome .
the coding regions of those homolog genes covered about  <dig>  mb of the black grouse draft genome.
we also checked how well each coding sequences of the chicken genes were covered, as this could infer the completeness of the annotated genes of the black grouse genome .
we found  <dig> genes, with a greater than 90% coverage of the coding regions.
this is, however, only a rough estimate, as the length of coding sequences could vary between black grouse genes and chicken genes.
we also noticed the interesting ‘u’ shape of the plot, that is, the coding regions of a large majority of the genes are either covered to a very large extent  or to a very small extent .
this may be explained if some genes that are highly divergent between black grouse and chicken could not be properly aligned in the reference guided assembly step.table  <dig>
number of genes from other bird genomes found to be homologous to the black grouse draft genome
histogram of the proportion of the coding regions covered of the annotated black grouse genes.
looking at the distribution of the annotated genes across the scaffolds, we found that the majority of the genes were identified on the  <dig> main chromosome scaffolds.
interestingly,  <dig> genes were identified from the unmapped contigs, suggesting that those genes could be not included in the reference chicken genome, or be highly divergent between black grouse and chicken.
the average gene density of the  <dig> main chromosome scaffolds was  <dig> e- <dig> gene/nucleotide.
chromosome  <dig> had the highest number of genes  as it was the longest chromosome.
chromosome  <dig> had the highest gene density of  <dig> e- <dig> gene/nucleotide, while chromosome z had the lowest gene density of  <dig> e- <dig> gene/nucleotide.
genomic repeats were identified using all known avian genomic repeats as references.
we found a total length of  <dig>  mb genomic repeats, which accounted for  <dig> % of the draft genome .
these numbers are lower than those of chicken and zebra finch, but similar to those of turkey  <cit> .
the chicken genome and the zebra finch genome were developed exclusively by sanger sequencing and have a better quality than that of turkey and black grouse.
this could be the reason why we found less genomic repeats in the black grouse draft genome.
the black grouse draft genome also had low numbers of simple repeat elements and low complexity regions compared to the other species.
however, this can be explained by the fragmented nature of the black grouse draft genome where simple repeats elements and low complexity regions may simply be represented by ‘n’ blocks, preventing the program from detecting them.table  <dig>
information on repeat elements identified from sequenced bird genomes
identification of snps
even though the genome sequence is only based on one individual, the fact that this wild, outbred bird was highly heterozygous allowed us to identify a large number of snps  <cit> .
to this end, we mapped all the filtered sequencing reads to the black grouse draft genome.
983 m reads, including the ones from the single-end library, the singletons from the mate-paired libraries and the paired reads of the two mate-paired libraries, were mapped to the genome with average coverage of  <dig> x. we set the coverage cut-off for the snp calling as  <dig> to 100x.
we only accepted snp sites with certain level of coverage to ensure the quality of the snp calling but also to avoid the sites with unusually high coverage, as they might be the result of incorrect mapping of reads from duplicated regions.
thus, we finally obtained  <dig> high quality snps,  <dig>  of which were from the coding regions .
the transition/transversion ratio of the snps was  <dig> .
the snp density of this black grouse individual was  <dig> %, which was higher than that of turkey   <cit> .
however, the individual used in the turkey genome sequencing project was inbred, whereas the black grouse individual we used was from a large outbred natural population.
interestingly, we found that the snp density of black grouse was close to that of the giant panda , which was inferred from a captive but outbred individual  <cit> .table  <dig>
number and density of single nucleotide polymorphisms  identified in the genome sequence from one outbred black grouse individual
we further investigated the snps on the  <dig> large chromosome scaffolds .
we classified the chromosomes into four categories: macro-chromosomes , intermediate-chromosomes , micro-chromosomes  and sex chromosome .
we found that the macro-chromosomes had the highest heterozygosity while the sex chromosome had the lowest.
the heterozygosity of the micro-chromosomes was also low.
this might be because that the micro-chromosomes have a higher gene density in the black grouse.
in contrast, the sex chromosome had the lowest density of genes but also had a low heterozygosity.
similar patterns have been observed in a wide variety of organisms and are explained by the fact that the effective population size of chromosome z is theoretically  <dig>  compared to that of the autosomes  <cit> .
in addition, the reduced variation on the z  could also be interpreted as the result of faster evolution and purifying selection .
comparative genomics
since the scaffolds of the black grouse draft genome were developed by using the chicken genome as reference, we could not investigate the genomic variations of black grouse, chicken and other species from a genomic rearrangement perspective, however, the sequences allowed us to conduct a comprehensive comparative genomic analysis at the level of nucleotide variation.
for this analysis, we focused on the main chromosomes  and examined the nucleotide divergence  between black grouse, chicken and turkey.
the downloaded chicken genome was split to  <dig> sequences, of which  <dig>  could be mapped to the black grouse main chromosome scaffolds .
this alignment covered 795 m  of the sequenced sites of the main black grouse chromosomes.
the downloaded turkey genome was split to  <dig> sequences, of which  <dig>  could be mapped to the main black grouse chromosome scaffolds.
this alignment covered 703 m  of the sequenced sites of the main black grouse chromosomes.
the turkey genome had a higher mapping percentage but a much lower coverage of the sequenced sites of the black grouse genome, as the turkey genome sequences were of lower quality  compared to those of chicken.
the average nucleotide divergence between the  <dig> black grouse and chicken chromosomes was  <dig>  ±  <dig> , with the divergence between black grouse and turkey was  <dig>  ±  <dig> .
those nucleotide divergence estimates were a little lower compared to the studies on chicken and turkey  <cit> , this, however, might be because we used a genome mapping approach to complete the alignment, which could make us miss the most highly divergent sequences.
the black grouse, the chicken and the turkey are closely related species.
counter to our findings here, phylogenetic analysis suggests that the black grouse is more closely related to turkey than chicken  <cit> .
this might be because, since we used chicken genome as reference to construct the black grouse draft genome for the heterozygous nucleotide sites, the choice of the nucleotides could be biased towards the chicken reference genome.
to further investigate the nucleotide divergence, we grouped the chromosomes into four categories  as described in the last section.
we found that the nucleotide divergence of intermediate-chromosomes was slightly lower than that of the macro-chromosomes, and the nucleotide divergence of micro-chromosomes was slightly higher than that of macro-chromosomes and intermediate-chromosomes .
the nucleotide divergence of the z chromosome was also higher than for the autosomes.
the observation of increased divergence rates on sex chromosomes is often referred to as the faster x effect  <cit> .
this pattern is generally thought to arise from the smaller effective population size of sex chromosome compared to autosomes, or an increased accumulation of recessive adaptive mutations  <cit> .figure  <dig>
nucleotide divergence of the chromosome groups.
macro-chromosome: chromosome 1 ~  <dig>  intermediate-chromosome: chromosome 6 ~  <dig>  micro-chromosome: chromosome 11 ~  <dig>  sex chromosome: chromosome z. ck represents the comparison between black grouse and chicken.
tk represents the comparison between black grouse and turkey.
finally, we calculated the nucleotide divergence by 50 kb sliding window size to screen for the highly divergent genome regions between black grouse and chicken and between black grouse and turkey.
a previous study suggested the divergent rate of the galliform mhc region was approximate  <dig>   <cit> .
here, we used a divergence rate of  <dig>  as the cut-off and identified  <dig> regions which had a high nucleotide divergence rate .
these regions could potentially harbour genes or gene regulatory elements which are important to some specific phenotypic attributes of the black grouse.
among the identified genomic regions,  <dig> contained genes or gene fragments and a total of  <dig> genes were localized in these high divergence regions.
those genes are important to the future in-depth studies of the lineage specific evolution of the black grouse.figure  <dig>
example of the identification of the highly divergent regions and the related genes.
the nucleotide divergence was calculated by 50 kb sliding window.
on chromosome  <dig>  the plotted dot ‘5650001-5700000’ of the sliding window was higher than  <dig> , so this region was identified as a highly divergent region.
below the genes located on this region were also identified.
CONCLUSIONS
in this study, using the chicken genome as a reference, we successfully assembled the whole draft genome of black grouse.
the draft genome consists of  <dig> scaffolds with a total length of 1022 mb , and additional  <dig> unscaffoled contigs with total length of  <dig>  mb.
this corresponds to a high coverage of the chicken chromosomes 1 ~  <dig> and chromosome z, with a total length of 1001 mb .
although the continuously sequenced blocks on the scaffolds are fragmented, the draft genome has a good coverage of the homologous chicken genes, and  <dig>  of the chicken genes were identified on the black grouse draft genome.
notably,  <dig> % of the coding regions of the homologous genes have more than 90% proportion of their sequences covered.
to our knowledge, this is the first time a large eukaryote genome was developed by solid short sequencing technology and reference guided assembly bioinformatic pipeline.
our study demonstrates how a moderate sequencing effort can be combined with existing genome references to accomplish a large genome project.
we identified a large number  of snps and identified the genomic regions we suggest are important for the lineage specific evolution of black grouse.
from the above analysis, we note that the sex chromosome  had lower reference assembly efficiency, lower snp density but a higher nucleotide divergence between black grouse and other galliform species.
those multiple evidences support the faster x  hypothesis of the sex chromosome, which states that the chromosome x  evolves faster than the autosomes due to its lower effective population size and recombination rate.
we also observed that microchromosome  <dig> which harbours the mhc region in galliforms was highly divergent among species which may indicate faster evolution in this genomic region.

 next generation sequencing has spurred a revolution in the development of genomic tools for non-model organisms. in particular, sequencing complete transcriptomes or complexity-reduced fractions of genomes has enabled the identification of genome-wide molecular markers such as single nucleotide polymorphisms and microsatellites. such investigations have also addressed fundamental questions in molecular ecology and evolution, such as the genomic basis for speciation, morphological variation, disease resistance and selection on life history traits. a complete genome sequence is the ultimate genomic tool for a species. if such a sequence is available it is possible to conduct large-scale, in-depth studies of many important molecular biology processes such as gene expression, transcription regulation, alternative splicing, epigenetic modifications and gene-protein interactions which are important in ecological studies. however, applying ngs technologies such as de-novo sequencing on a large eukaryotic genome is still rare, as it represents a considerable investment. the sheer volume of data generated and the computational facilities needed to assemble and analyse it may limit the number of non-specialized labs that are currently able to embark on such a project. however, more whole genome studies are needed to address fundamental questions on the evolution of genome organisation, such as which regions are conserved and which regions change when taxa diverge and become separate species. published whole ngs genomes of non-model organisms include giant panda, cod, naked mole rat, macaque, tasmanian devil, budgerigar, puerto rican parrot, heliconius butterfly, aye-aye, collared flycatcher, as well as the mammalian genomes recently sequenced at the broad institute. the large number of publically available whole genome sequences from both model and non-model organisms can be used to aid genomic investigations in related organisms. one approach is to directly transfer the genomic resources from a model organism to the study species, which would then be called ‘genome enabled taxa’. this strategy has been used successfully to develop resources such as microsatellite markers, snps, microarrays and exon capture arrays. alternatively, the genome sequence from a related model organism can be used in the assembly of short read data from the focal species, a process known as reference guided assembly. here, we describe a reference guided, whole genome assembly of the black grouse. we take advantage of the close relationship between this species and the well characterized chicken and develop a reference guided assembly pipeline to construct a draft genomic sequence. the black grouse is well studied as a model for ecology and conservation biology but, until recently, genomic resources for this species have been largely lacking. this work completes our genomic tools development, previously initiated by characterizing the transcriptome and sequencing the major histocompatibility complex region in this species.figure male black grouse displaying at a lek. in theory there are two different ways of using the reference sequence to guide the assembly process. under an “align-then-assemble” strategy, the reads are first mapped to the reference sequence and clusters of reads mapping to the same location are then extracted and assembled de-novo. alternatively, in the “assemble-then-align” strategy, the reads are first de-novo assembled and the resulting contigs are then aligned to the reference genome to close gaps and create scaffolds. in our reference guided assembly pipeline we use a combination of these two approaches by mapping both mate-paired reads and de-novo assembled contigs to the reference genome and combining these alignments to produce the final scaffolds. this approach has some similarities to the previously published “reference-assisted chromosome assembly”. importantly, we also demonstrate the utility of the solid sequencing technology for whole genome de-novo sequencing. due to the short reads produced by this method compared to more widely used and hiseq sequencers, the solid platform has not been used before for sequencing of vertebrate sized genomes in a non-model organism. bacterial and fungal genomes have, however, previously been sequenced solely based on this technology. even though the de-novo assembly of our short read data was fragmented we were still able to successfully cover a large proportion of the genome using our reference guided approach. we used the draft genome both to identify a vast number of snps and to perform comparative genomic analyses.figure flow chart of our reference guided genome assembly pipeline. all reads were first de-novo assembled. in the second step both long contigs and original mate-paired reads were mapped to the chicken reference genome and merged to produce the black grouse backbone scaffolds. finally gap-filling was performed by mapping all contigs back to the backbone scaffolds, producing the draft genome assembly. in addition all contigs not mapping to the backbone scaffolds were added to the assembly after removing sequences arising from possible contamination using a blast approach. for more details about the procedures please see the methods section. in the figures to the right the chicken reference genome is indicated by the blue line while reads, contigs and scaffolds from the black grouse are shown in red. the light red parts of the final scaffold indicate regions with gaps in the black grouse sequence. within brackets in the boxes to the left are the software used for each different stage of the assembly process.  solid sequencing the raw sequencing data was comprised of 793 m reads with a read length of 75 bp which were generated for the single-end library, 1642 m reads with read length of 60 bp × 60 bp which were generated for the 2 kb mate-paired library, and 1548 m reads with read length of 60 bp × 60 bp which were generated for the 5 kb mate-paired library. the raw reads are deposited in the ncbi sequence read archive under the accession number sra after quality and length filtering, 423 m reads were retained for the single-end library, 320 m of which were 75 bp in length. for the 2 kb mate-paired library, 857 m reads were retained after filtering, and 663 m of them were 60 bp in length. for those filtered reads, 519 m were properly paired, and the rest were only retained as unpaired reads. for the 5 kb mate-paired library, 847 m reads were retained after filtering, 648 m of which were of 60 bp in length. for those filtered reads, 520 m were properly paired, and the rest were only retained as unpaired reads. therefore, 2127 m high quality sequencing reads with the total length of approximately 133 gb were kept in downstream analysis. if we assume that the genome size of black grouse is similar to that of chicken, the estimated mean sequencing coverage of the black grouse genome was 127x. reference guided assembly the reference guided assembly is comprised of several steps, including de novo assembly, reference mapping and the merging of these results. in the first step, all the 2127 m filtered high quality reads were de novo assembled by soapdenovo. we were able to generate preliminary contigs with a total length of 937 mb. as expected, the de novo assembly was more fragmented compared to some other studies which also used short-read sequencing technologies, this is because in this study we only had three sequencing libraries with a maximum insert size of 5 kbp and the sequencing reads produced by the solid technology were relatively short. the solid platform is believed to produce high quality reads. all the filtered data we used in our analyses had an error rate not larger than %. however, the short read length seriously affects its performance in pure de novo assembly. longer sequencing reads produced by platforms such as ion-torrent or pacbio usually produce larger contigs and such data could be used to improve our assembly in the future. in the next step, we aligned all long contigs to the chicken genome and were able to map of them. the total mapped length was 438 mb. at the same time, we also aligned the filtered and properly paired reads from the mate-paired libraries to the chicken genome resulting in 451 m successfully mapped reads. these two sets of mapped reads were merged and this resulted in a 805 mb black grouse genome backbone scaffold. finally, we mapped the de novo assembled contigs back to the black grouse backbone scaffolds and had of them mapped. therefore, we succeeded to cover 833 mb of the 1046 mb chicken genome, and of the chicken scaffolds. we covered 826 mb of the 1001 mb main chicken chromosomes. in addition, we also retained unmapped contigs with a total length of  mb. the resulting black grouse draft genome assembly consisted of scaffolds with a total length of 1022 mb. the genome assembly is deposited in the ncbi whole genome shotgun database under the submission number jdsl among the scaffolds, the largest, corresponding to the chicken chromosomes 1 ~  and chromosome z, had a total length of 1001 mb. the average coverage of the chromosomes was %. however, this coverage was not distributed evenly across the chromosome scaffolds or across the chromosomal regions. we noticed that chromosome chromosome chromosome and chromosome z were not well covered. chromosome z is the avian sex chromosome and chromosome harbours the mhc genes. these might be more divergent between black grouse and chicken than the rest of the genome, which may have led to the poor assembly. for chromosome an additional reason might be that the chicken assembly of this chromosome is still not perfect and contain large n chunks. furthermore, we have previously shown that when comparing this region among different galliform species, there are several gene copy divergences as well as genomic inversions in the mhc region on chromosome. we further examined the quality of the black grouse draft genome, and found that although the sequence coverage was high, the scaffold sequences were highly fragmented. if the draft genome is split at all ‘n’ sites present on the scaffolds, it has continuous sequenced blocks. this is not unexpected since the solid data was short and we used a reference guided approach to produce the draft genome, and the existing bioinformatic tools available are not mature enough in dealing with this strategy. the samtools pipeline we used generates consensus sequences solely based on the coordinates of the reference genome, which might introduce a number of additional ‘n’s in the resulted sequences. in addition, a number of the long ‘n’ chunks are also present in the reference chicken genome, and might thereby be introduced into the black grouse draft genome through the reference guided assembly process.figure heatmap showing the proportion of the regions sequenced on the chromosome scaffolds. distributions of the continuous sequence-blocks and the interleaved ‘n’ blocks. the blue lines indicate the total length of the respective blocks. the sequenced blocks larger than 10000 bp and the ‘n’ blocks larger than 30000 bp were binned at the end of the axis. annotation the fragmented state of the draft genome limited our ability to systematically perform ab-initio predictions of genes or genomic repeats. instead we used comparative methods to identify the gene regions and the genomic repetitive regions. from the reciprocal blast result, we found that chicken genes had homologs on the black grouse genome. the coding regions of those homolog genes covered about  mb of the black grouse draft genome. we also checked how well each coding sequences of the chicken genes were covered, as this could infer the completeness of the annotated genes of the black grouse genome. we found genes, with a greater than 90% coverage of the coding regions. this is, however, only a rough estimate, as the length of coding sequences could vary between black grouse genes and chicken genes. we also noticed the interesting ‘u’ shape of the plot, that is, the coding regions of a large majority of the genes are either covered to a very large extent or to a very small extent. this may be explained if some genes that are highly divergent between black grouse and chicken could not be properly aligned in the reference guided assembly step.table number of genes from other bird genomes found to be homologous to the black grouse draft genome histogram of the proportion of the coding regions covered of the annotated black grouse genes. looking at the distribution of the annotated genes across the scaffolds, we found that the majority of the genes were identified on the main chromosome scaffolds. interestingly, genes were identified from the unmapped contigs, suggesting that those genes could be not included in the reference chicken genome, or be highly divergent between black grouse and chicken. the average gene density of the main chromosome scaffolds was e- gene/nucleotide. chromosome had the highest number of genes as it was the longest chromosome. chromosome had the highest gene density of e- gene/nucleotide, while chromosome z had the lowest gene density of e- gene/nucleotide. genomic repeats were identified using all known avian genomic repeats as references. we found a total length of  mb genomic repeats, which accounted for % of the draft genome. these numbers are lower than those of chicken and zebra finch, but similar to those of turkey. the chicken genome and the zebra finch genome were developed exclusively by sanger sequencing and have a better quality than that of turkey and black grouse. this could be the reason why we found less genomic repeats in the black grouse draft genome. the black grouse draft genome also had low numbers of simple repeat elements and low complexity regions compared to the other species. however, this can be explained by the fragmented nature of the black grouse draft genome where simple repeats elements and low complexity regions may simply be represented by ‘n’ blocks, preventing the program from detecting them.table information on repeat elements identified from sequenced bird genomes identification of snps even though the genome sequence is only based on one individual, the fact that this wild, outbred bird was highly heterozygous allowed us to identify a large number of snps. to this end, we mapped all the filtered sequencing reads to the black grouse draft genome. 983 m reads, including the ones from the single-end library, the singletons from the mate-paired libraries and the paired reads of the two mate-paired libraries, were mapped to the genome with average coverage of x. we set the coverage cut-off for the snp calling as to 100x. we only accepted snp sites with certain level of coverage to ensure the quality of the snp calling but also to avoid the sites with unusually high coverage, as they might be the result of incorrect mapping of reads from duplicated regions. thus, we finally obtained high quality snps, of which were from the coding regions. the transition/transversion ratio of the snps was. the snp density of this black grouse individual was %, which was higher than that of turkey. however, the individual used in the turkey genome sequencing project was inbred, whereas the black grouse individual we used was from a large outbred natural population. interestingly, we found that the snp density of black grouse was close to that of the giant panda, which was inferred from a captive but outbred individual.table number and density of single nucleotide polymorphisms identified in the genome sequence from one outbred black grouse individual we further investigated the snps on the large chromosome scaffolds. we classified the chromosomes into four categories: macro-chromosomes, intermediate-chromosomes, micro-chromosomes and sex chromosome. we found that the macro-chromosomes had the highest heterozygosity while the sex chromosome had the lowest. the heterozygosity of the micro-chromosomes was also low. this might be because that the micro-chromosomes have a higher gene density in the black grouse. in contrast, the sex chromosome had the lowest density of genes but also had a low heterozygosity. similar patterns have been observed in a wide variety of organisms and are explained by the fact that the effective population size of chromosome z is theoretically compared to that of the autosomes. in addition, the reduced variation on the z could also be interpreted as the result of faster evolution and purifying selection. comparative genomics since the scaffolds of the black grouse draft genome were developed by using the chicken genome as reference, we could not investigate the genomic variations of black grouse, chicken and other species from a genomic rearrangement perspective, however, the sequences allowed us to conduct a comprehensive comparative genomic analysis at the level of nucleotide variation. for this analysis, we focused on the main chromosomes and examined the nucleotide divergence between black grouse, chicken and turkey. the downloaded chicken genome was split to sequences, of which could be mapped to the black grouse main chromosome scaffolds. this alignment covered 795 m of the sequenced sites of the main black grouse chromosomes. the downloaded turkey genome was split to sequences, of which could be mapped to the main black grouse chromosome scaffolds. this alignment covered 703 m of the sequenced sites of the main black grouse chromosomes. the turkey genome had a higher mapping percentage but a much lower coverage of the sequenced sites of the black grouse genome, as the turkey genome sequences were of lower quality compared to those of chicken. the average nucleotide divergence between the black grouse and chicken chromosomes was  ± , with the divergence between black grouse and turkey was  ± . those nucleotide divergence estimates were a little lower compared to the studies on chicken and turkey, this, however, might be because we used a genome mapping approach to complete the alignment, which could make us miss the most highly divergent sequences. the black grouse, the chicken and the turkey are closely related species. counter to our findings here, phylogenetic analysis suggests that the black grouse is more closely related to turkey than chicken. this might be because, since we used chicken genome as reference to construct the black grouse draft genome for the heterozygous nucleotide sites, the choice of the nucleotides could be biased towards the chicken reference genome. to further investigate the nucleotide divergence, we grouped the chromosomes into four categories as described in the last section. we found that the nucleotide divergence of intermediate-chromosomes was slightly lower than that of the macro-chromosomes, and the nucleotide divergence of micro-chromosomes was slightly higher than that of macro-chromosomes and intermediate-chromosomes. the nucleotide divergence of the z chromosome was also higher than for the autosomes. the observation of increased divergence rates on sex chromosomes is often referred to as the faster x effect. this pattern is generally thought to arise from the smaller effective population size of sex chromosome compared to autosomes, or an increased accumulation of recessive adaptive mutations.figure nucleotide divergence of the chromosome groups. macro-chromosome: chromosome 1 ~  intermediate-chromosome: chromosome 6 ~  micro-chromosome: chromosome 11 ~  sex chromosome: chromosome z. ck represents the comparison between black grouse and chicken. tk represents the comparison between black grouse and turkey. finally, we calculated the nucleotide divergence by 50 kb sliding window size to screen for the highly divergent genome regions between black grouse and chicken and between black grouse and turkey. a previous study suggested the divergent rate of the galliform mhc region was approximate. here, we used a divergence rate of as the cut-off and identified regions which had a high nucleotide divergence rate. these regions could potentially harbour genes or gene regulatory elements which are important to some specific phenotypic attributes of the black grouse. among the identified genomic regions, contained genes or gene fragments and a total of genes were localized in these high divergence regions. those genes are important to the future in-depth studies of the lineage specific evolution of the black grouse.figure example of the identification of the highly divergent regions and the related genes. the nucleotide divergence was calculated by 50 kb sliding window. on chromosome the plotted dot ‘5650001-5700000’ of the sliding window was higher than, so this region was identified as a highly divergent region. below the genes located on this region were also identified.  in this study, using the chicken genome as a reference, we successfully assembled the whole draft genome of black grouse. the draft genome consists of scaffolds with a total length of 1022 mb, and additional unscaffoled contigs with total length of  mb. this corresponds to a high coverage of the chicken chromosomes 1 ~  and chromosome z, with a total length of 1001 mb. although the continuously sequenced blocks on the scaffolds are fragmented, the draft genome has a good coverage of the homologous chicken genes, and of the chicken genes were identified on the black grouse draft genome. notably, % of the coding regions of the homologous genes have more than 90% proportion of their sequences covered. to our knowledge, this is the first time a large eukaryote genome was developed by solid short sequencing technology and reference guided assembly bioinformatic pipeline. our study demonstrates how a moderate sequencing effort can be combined with existing genome references to accomplish a large genome project. we identified a large number of snps and identified the genomic regions we suggest are important for the lineage specific evolution of black grouse. from the above analysis, we note that the sex chromosome had lower reference assembly efficiency, lower snp density but a higher nucleotide divergence between black grouse and other galliform species. those multiple evidences support the faster x hypothesis of the sex chromosome, which states that the chromosome x evolves faster than the autosomes due to its lower effective population size and recombination rate. we also observed that microchromosome which harbours the mhc region in galliforms was highly divergent among species which may indicate faster evolution in this genomic region. 
our study demonstrates how a moderate sequencing effort can be combined with existing genome references to generate a draft genome for a non-model species. for example, comparative genomic studies have suggested that the sex chromosomes and the regions harbouring the immune defence genes in the major histocompatability complex may evolve faster than other genomic regions. our results support the hypothesis that the chromosome x evolves faster than the autosomes and our data are consistent with the mhc regions being more liable to change than the genome average. in this study, we present the draft genome of the black grouse, which was developed using a reference-guided assembly strategy. the draft genome well covers the main chicken chromosomes 1 ~  and z which have a total length of 1001 mb. the draft genome is fragmented, but has a good coverage of the homologous chicken genes. we generated 133 gbp of sequence data from one black grouse individual by the solid platform and used a combination of de novo assembly and chicken reference genome mapping to assemble the reads into scaffolds with a total length of 1022 mb. in addition, we identified ~1 m snps from the genome and identified genomic regions which had a high nucleotide divergence between black grouse and chicken or between black grouse and turkey. especially, % of the coding regions of the homologous genes have more than 90% proportion of their sequences covered. the advent of the next generation sequencing technologies has made it possible to study which genomic regions are evolutionary liable to change and which are static, as well as enabling an increasing number of genome studies of non-model species.
 the different regions of a genome do not evolve at the same rate. for example, comparative genomic studies have suggested that the sex chromosomes and the regions harbouring the immune defence genes in the major histocompatability complex may evolve faster than other genomic regions. the advent of the next generation sequencing technologies has made it possible to study which genomic regions are evolutionary liable to change and which are static, as well as enabling an increasing number of genome studies of non-model species. however, de novo sequencing of the whole genome of an organism remains non-trivial. in this study, we present the draft genome of the black grouse, which was developed using a reference-guided assembly strategy.  we generated 133 gbp of sequence data from one black grouse individual by the solid platform and used a combination of de novo assembly and chicken reference genome mapping to assemble the reads into scaffolds with a total length of 1022 mb. the draft genome well covers the main chicken chromosomes 1 ~  and z which have a total length of 1001 mb. the draft genome is fragmented, but has a good coverage of the homologous chicken genes. especially, % of the coding regions of the homologous genes have more than 90% proportion of their sequences covered. in addition, we identified ~1 m snps from the genome and identified genomic regions which had a high nucleotide divergence between black grouse and chicken or between black grouse and turkey.  our results support the hypothesis that the chromosome x evolves faster than the autosomes and our data are consistent with the mhc regions being more liable to change than the genome average. our study demonstrates how a moderate sequencing effort can be combined with existing genome references to generate a draft genome for a non-model species. electronic supplementary material the online version of this article contains supplementary material, which is available to authorized users. issue-copyright-statement© the author 2014 