BACKGROUND
four remarkable evolutionary phenomena are associated with organellar genomes of geraniaceae.
first, mitochondrial genomes show multiple, major shifts in rates of synonymous substitutions, especially in the genus pelargonium <cit> .
rate fluctuations of such magnitude have been documented in only two other plant lineages, plantago <cit>  and silene <cit> .
second, mitochondrial genomes have experienced extensive loss of genes and sites of rna editing.
at least  <dig> putative gene losses have been documented in erodium <cit> , and mitochondrial genes sequenced from pelargonium x hortorum had a drastic reduction in predicted or verified rna editing sites compared to all other angiosperms examined  <cit> .
third, genome-wide comparisons of nucleotide substitutions in plastid dna indicated rapid rate acceleration in genes encoding ribosomal proteins, rna polymerase, and atp synthase subunits in some lineages.
in the case of rna polymerase genes there was evidence for positive selection  <cit> .
fourth, plastid genomes of geraniaceae are the most highly rearranged of any photosynthetic land plants examined  <cit> .
multiple and extreme contractions and expansions of the inverted repeat  have resulted in genomes with both the largest ir  as well as the complete loss of this feature  <cit> .
considerable accumulation of dispersed repeats associated with changes in gene order has been documented along with disruption of highly conserved operons and repeated losses and duplications of genes  <cit> .
in p. x hortorum plastids, these genomic changes have generated several fragmented and highly divergent rpoa-like orfs of questionable functionality  <cit> , despite the fact that rpoa encodes an essential component of the plastid-encoded rna polymerase .
because nuclear genes supply both organelles with the majority of their proteins, it is likely that the extensive organellar genomic upheaval in geraniaceae will also influence the evolution of organelle-targeted genes in the nuclear genome.
for example, given the drastic reduction of rna editing in geraniaceae mitochondrial transcripts, it is reasonable to expect a correlated reduction of nucleus-encoded pentatricopeptide repeat  proteins, many of which are critical for organellar rna editing  <cit> .
the uncertain status of the p. x hortorum plastid-encoded rpoa gene is also likely to have nuclear consequences.
if this plastid gene is not functional, then a functional copy might have been relocated to the nuclear genome, which has only occurred once in the evolution of land plants in mosses  <cit> .
alternatively, it is possible that pep has become nonfunctional in p. x hortorum, as observed in the holoparasite phelipanche aegyptiaca <cit> .
in p. aegyptiaca, loss of all plastid-encoded pep components  resulted in the parallel loss of the requisite nucleus-encoded components  that assemble with the plastid encoded proteins to form the core of the pep holoenzyme  <cit> .
in contrast, if the highly divergent plastid rpoa gene is still functional in p. x hortorum, then the typical set of sigma factors should be present in the nuclear genome.
one prerequisite to begin to address the effects of organellar genomic upheaval on the nuclear genome in geraniaceae, is availability of nuclear sequence information.
transcriptome sequencing provides a tractable proxy for nuclear gene space.
the use of next-generation sequencing  for transcriptome sequencing is widespread because volumes of data can be generated rapidly at a low cost relative to traditional sanger sequencing  <cit> .
the assembly of reads into contigs may be executed using a de novo or a reference-based approach  <cit> .
in studies of non-model organisms, de novo assembly is more commonly used due to the absence of a closely related reference  <cit> .
a survey of recent transcriptome studies in comparative biology demonstrates that most sequencing projects are focusing on non-model organisms where little or no genomic data is available  <cit> .
the lack of a reference genome makes the reconstruction and evaluation of the transcriptome assembly challenging.
several issues must be addressed when performing transcriptome sequencing of non-model organisms, including which ngs platform should be employed, how much sequence data is needed to provide a comprehensive transcriptome, which assembler should be utilized, and what tissues should be sampled.
this paper provides a comprehensive comparison of the transcriptomes of two non-model plant species, pelargonium x hortorum and geranium maderense, from the two largest genera of geraniaceae.
there were three primary goals for the initial comparative transcriptome analysis in geraniaceae:  what are the best sequencing platforms and assembly methods for generating a high-quality transcriptome that broadly covers gene space in the absence of a reference genome?
does sequencing from multiple tissue types improve the breadth of transcriptome coverage?
are there any losses of ppr proteins involved in rna editing and sigma factors associated with pep in geraniaceae?
RESULTS
ribosomal rna content and illumina library complexity
to assess the efficiency of ribosomal rna  depletion in geraniaceae transcriptome libraries rrna contigs were identified using rrna from arabidopsis thaliana as a reference.
all illumina reads  were mapped to rrna contigs as described in methods, and  <dig> % and 2% of the reads of g. maderense and p. x hortorum were identified as rrna reads, respectively.
library complexity was analyzed using picard  <cit>  and rrna reads were eliminated prior to the analysis.
the percentages of unique start sites were  <dig> % and  <dig> % for g. maderense and p. x hortorum, respectively.
the values for rrna content and library complexity were comparable to other transcriptome analyses using similar approaches  <cit> .
assessment of sequencing platforms and assemblers for transcriptome assembly
to determine the optimal sequencing and assembly strategy, the efficacy of five different assemblers was examined using two initial data sets generated by roche/ <dig> flx and illumina hiseq  <dig> platforms for p. x hortorum.
the illumina run produced approximately  <dig> times more sequence data than the  <dig> run, even though the cost of the  <dig> data was at least four times more than the illumina data .
a comparison of basic assembly statistics  showed that the trinity assembler outperformed all other platform/software combinations in terms of number of contigs, number of assembled nucleotides, mean and maximum contig length, and n <dig>  more generally, the illumina assemblers consistently outperformed the  <dig> assemblers, although the mira and newbler  <dig> assemblers produced longer maximal contigs than soapdenovo and soapdenovo-trans .
to determine the amount of usable protein sequence information generated by each assembler, the assemblies were translated as described in methods and compared .
again, the illumina assemblers outperformed the  <dig> assemblers in all metrics, with the trinity assembler providing the most amino acids with the longest mean and maximal sequences.
the length distribution of assembled nucleotides and translated amino acids further confirms that trinity outperformed soapdenovo and soaptrans, and all three illumina assemblers outperformed the  <dig> assemblers .
pelargonium
x
hortorum
transcriptome dataset read statistics
x
hortorum
transcriptome
x
hortorum
two important considerations in assembly analysis are the breadth of gene space coverage and the degree of coverage fragmentation.
a good assembler should generate high-quality assemblies that contain as many reference transcripts as possible, and each reference transcript should be covered as completely as possible with a single long contig rather than a combination of several short contigs.
to assess assembly coverage and fragmentation, two published data bases were used,  <dig> ultra-conserved ortholog  coding sequence  <cit>  from arabidopsis and  <dig> single copy nuclear genes shared between arabidopsis, oryza, populus, and vitis <cit> .
trinity and soaptrans outperformed all other assemblers in terms of the percentage of reference genes identified, completeness of coverage , and contiguity of coverage , with trinity performance slightly better than soaptrans at higher thresholds .
to examine whether the superior performance of trinity and soaptrans was due to the much larger amount  of illumina data than  <dig> data, the illumina assemblers were re-anlayzed using a data set containing 1/40th of the illumina reads .
in terms of contiguity and completeness, the performance of trinity using the reduced illumina data set remained superior to the  <dig> programs  that used the entire  <dig> data sets.
in contrast, the performance of soapdenovo and soaptrans were noticeably worse with the reduced illumina data set than with the full data set, producing results that were generally worse than the original  <dig> assemblies.
effect of sequencing depth on assembly coverage breadth and fragmentation
to determine how much sequence data is needed to assemble a high-quality transcriptome with broad coverage,  <dig> , <dig> reads for g. maderense and  <dig> , <dig> reads for p. x hortorum were generated on the illumina hiseq  <dig> platform assembled using trinity with different increments of reads from 5% to 100% of the total.
while the number of contigs assembled continued to increase with increasing numbers of reads , the percentage of reference genes recovered and their contiguity and completeness plateaued at approximately 40% of the total reads .
including the remaining 60% of the reads increased contiguity and completeness by only 1% to 2% .
although there were more translated contigs of g. maderense than p. x hortorum, the contiguity and completeness of both species were very similar.
although increasing the number of reads beyond 10% contributed little to finding novel hits to the local arabidopsis data base, increasing the amount of data did help extend the existing contigs and generate longer alignments to reference genes.
to evaluate this, the contiguity of all contigs relative to the two published databases was calculated at different contiguity thresholds up to 100% .
the inclusion of more reads generated assemblies with higher contiguity, especially when contiguity thresholds were greater than 50%.
to allow for the high level of sequence divergence between geraniaceae and arabidopsis, the number of contigs that had contiguity thresholds ≥80% was calculated.
when 100% of the reads were used  <dig> contigs and  <dig> contigs were found in g. maderense and p. x hortorum, respectively.
reducing the read input to 40% reduced contiguity values by 7%  in g. maderense and 11%  in p. x hortorum.
functional assessment of geraniaceae nuclear transcriptomes
the assemblies generated using 100% of the reads for both geraniaceae species were used for functional annotation.
assemblies were first aligned against the ncbi nr database and the alignment results were used to generate the gene ontology  terms.
of the  <dig>  contigs in p. x hortorum,  <dig>   had blast hits;  <dig>   were annotated and  <dig>  go terms were retrieved .
of the  <dig>  contigs in g. maderense,  <dig>   had blast hits;  <dig>   were annotated  and  <dig>  go terms were retrieved.
the annotation files are shown in additional file  <dig>  the distribution of gene ontology annotations was examined using go-slim  ontology to compare the transcriptomes of g. maderense and p. x hortorum.
although the number of annotated contigs differed substantially between the two transcriptomes , the proportion of annotated contigs in all categories with >1% representation within the categories cellular component, molecular function, and biological process were very similar .
this similarity persists even though only emergent leaves were sampled for g. maderense versus four tissue types  for p. x hortorum.
geranium maderense
and
pelargonium
x
hortorum
go = gene ontology; ec = enzyme code.
to more directly address the question whether sequencing from multiple tissue types improves the breadth of transcriptome coverage, orthologous genes between g. maderense and a. thaliana and between p. x hortorum and a. thaliana were identified.
of the  <dig>  protein sequences from a. thaliana, the g. maderense assembly had homologs to  <dig>  sequences and the p. x hortorum assembly had homologs to  <dig>  sequences.
the comparable numbers of orthologous genes found for the two geraniaceae species indicated that there was little improvement on the breadth of transcriptome coverage by sequencing from multiple tissue types .
identification of selected organelle targeted genes
pentatricopeptide repeat proteins  are a large family of rna binding proteins encoded by over  <dig> genes in angiosperms; most are organelle targeted and involved in regulating organelle gene expression.
the transcriptomes of p. x hortorum and g. maderense were annotated using  <dig> arabidopsis ppr sequences as a reference database .
the overall number of ppr genes varied considerably between the two geraniaceae and arabidopsis, with ppr gene number reduced in p. x hortorum.
the numbers of p class ppr genes were found to be similar in all three species, whereas many fewer pls class genes were found in the geraniaceae, especially in p. x hortorum.
a
and sigma factor
b
distribution
arabidopsis thaliana
geranium maderense
pelargonium
x
hortorum
appr protein data of arabidopsis are from small and peeters  <cit> .
the ppr class represents the number contigs longer than  <dig> aa, which is the minimum length of ppr proteins identified in arabidopsis.
bthe number of total contigs and the number of intact contigs aligned to the reference sigma factors are shown.
intact contigs are those with start/stop codons on 5′ and 3′ ends, and without any internal stop codons.
sigma factors are nuclear encoded, plastid targeted proteins that assemble with four plastid encoded proteins  to form the core of the pep holoenzyme.
at least one copy of each of the six arabidopsis sigma factors was detected in both the g. maderense and p. x hortorum transcriptomes .
the nucleotide and amino acid sequence identities between arabidopsis/geranium and arabidopsis/pelargonium for all six sigma factors were very similar .
the four contigs from g. maderense that aligned to sigma factor  <dig> were similar to each other in nucleotide sequence identity , suggesting that they may represent variant copies of the same gene.
two of the three contigs from g. maderense that aligned to sigma factor  <dig> were very similar to each other but less so to the third contig .
sigma factors  <dig> and  <dig> were each represented by two p. x hortorum contigs, however only one of the contigs for each sigma factor appeared functional having start/stop codons at the 5′ and 3′ ends and lacking internal stop codons.
further experiments are needed to determine if the copies with internal stop codons are pseudogenes or assembly artifacts.
arabidopsis thaliana
sigma factors
arabidopsis thaliana
geranium maderense
pelargonium
x
hortorum
sequence identity
a
ain cases where there is more than one intact contig for a sigma factor, the one with highest sequence identity to arabidopsis was selected for comparison.
discussion
strategies for de novo assembly of transcriptomes
the use of ngs platforms is widespread and is applied in many research fields as volumes of data can be generated rapidly at a low cost relative to traditional sanger sequencing  <cit> .
rna-seq, one popular ngs application, provides an efficient and cost-effective way of obtaining transcriptome data.
there are a number of platforms available for generating ngs data  <cit> .
currently among the most popular are the roche/ <dig> flx  and the illumina hiseq  <dig>  platforms.
the roche/ <dig> flx system is advantageous when longer reads are important , whereas the illumina system provides deeper sequencing coverage at a reduced cost per base, albeit with shorter read length .
for each platform various assemblers have emerged but during the past several years roche  <dig> sequencing and the platform-specific assembler newbler has been the most common approach for de novo assembly of transcriptome data  <cit> .
this may be attributed to the idea that longer reads are more likely to overcome the specific challenges of de novo transcriptome assembly.
illumina sequencing has been used mainly when a related organism’s genome was available for reference-based assembly  <cit> , although due to recently increased read length it is becoming more common for use in de novo assembly as well  <cit> .
several recent studies compared the performance of different sequencing platforms and assembly methods  <cit>  but none of these comparisons evaluated the level of completeness or contiguity of their assemblies, nor was the performance of the assemblers evaluated without known genome information, which is the situation for any project on non-model organisms.
our comparisons of sequencing platforms and assemblers for the geraniaceae clearly indicated that the illumina platform with trinity assembly delivered the best performance in assembling a complete transcriptome in the absence of a reference genome.
the illumina assemblers  generated more contigs containing a greater total number of bases than the roche/ <dig> flx assemblers .
hile the mira assembly generated many more long contigs  than soapdenovo, the trinity assembly out-performed all others in delivering long contigs, suggesting that the trinity assembly contained more useful information than any of the other assemblies analyzed.
while the roche/ <dig> flx assemblies and the illumina soapdenovo assembly produced similar results with regard to completeness and contiguity, the illumina trinity and soaptrans assemblies obtained much higher values for both parameters indicating that these assemblies comprise many more nearly complete transcripts .
functional annotation of geraniaceae transcriptomes
a total of  <dig>   and  <dig>   contigs were annotated from g. maderense and p. x hortorum, respectively.
the low percentage of annotated contigs is most likely due to the large number of total contigs assembled.
the number of aligned and annotated contigs is comparable to nine other recently published transcriptomes  <cit> .
the number of annotated contigs in assemblies from both geraniaceae species was very similar for the three major categories cellular component, molecular function, and biological process .
this is encouraging since different tissues were sampled for the two species; only one tissue, emergent leaves for geranium and four tissues, emergent leaves, expanded leaves, roots and flowers for pelargonium.
particularly noteworthy is the detection of genes associated with flower and embryo development and pollen-pistil interaction since flowers were not sampled for geranium.
overall, this comparison indicates that there is no marked improvement in transcriptome breadth of coverage when sampling four tissues compared to only emergent leaves.
ppr proteins and sigma factors in geraniaceae
pprs are a large family of rna binding proteins encoded by over  <dig> genes in sequenced angiosperms.
most are organelle targeted and involved in regulating organelle gene expression  <cit> .
of the two classes  within the ppr family, those from pls class  have been reported to be involved in rna editing  <cit> .
previous studies have demonstrated correlated evolution of pls genes and rna editing sites in plants  <cit> .
consistent with these results, a reduction in pls genes  in geraniaceae was detected, where reduced editing frequency was previously demonstrated  <cit> .
the reduced editing frequency and reduced ppr content in geraniaceae is especially intriguing with respect to the increased mitochondrial substitution rate in this family.
although an inverse correlation between editing frequency and substitution rate has been noted previously in geraniaceae and other taxa  <cit> , the finding that ppr gene content is also reduced in geraniaceae indicates that this family is ideally suited for future studies assessing the evolutionary dynamics of editing frequency, ppr content, and mitochondrial substitution rates.
one long-standing question regarding the plastid genomes in geraniaceae is the putative loss of the rpoa gene from p. x hortorum <cit> .
the complete plastid genome sequence of this species revealed several rpoa-like open reading frames  that are highly divergent relative to rpoa genes in other angiosperms or even other geraniaceae  <cit> .
two alternative explanations were suggested for these observations:  a copy of the gene in the nucleus had gained functionality; or  at least one of the highly divergent rpoa-like orfs remains functional.
extensive evolutionary rate comparisons of plastid genes across the geraniaceae revealed that the other three pep subunits  have significantly elevated nucleotide substitution rates and have likely experienced positive selection  <cit> .
despite exhaustive searching of the nuclear transcriptome of p. x hortorum no copy of the rpoa gene was detected.
however, intact copies of all six sigma factors, which are required for pep to function  <cit> , were identified in the transcriptome.
the holoparasite phelipanche aegyptiaca lacks a functional pep and mining unigene files published in a recent transcriptomic study of parasitic plants  <cit>  failed to uncover a single sigma factor suggesting that in species where pep sequences are lost from the plastid the requisite sigma factors are also absent from the nuclear transcriptome.
the identification of all six sigma factors in the p. x hortorum transcriptome supports the likelihood that pep is active in p. x hortorum plastids.
CONCLUSIONS
with the widespread application of ngs techniques, the ability to process and analyze massive quantities of sequence data in a timely manner becomes imperative to a successful project.
regardless of the goals of a particular project, it is desirable to obtain data that is as accurate and complete as possible in a way that is cost effective as well as timely.
in this study a cross-platform comparison of de novo transcriptome assembly was conducted using representative species from the two largest genera of geraniaceae, g. maderense and p. x hortorum.
as no reference genome is available for geraniaceae, or any of its close relatives, this approach represents a truly de novo assembly allowing evaluation of efficacy among the platforms/assemblers that more closely resembles current ngs research.
the assembly of illumina hiseq  <dig> reads with trinity or soaptrans was highly effective in reconstructing, as completely as is currently feasible, the protein-coding transcripts of geraniaceae.
as for the differences between the two assemblers, trinity generated slightly more single contiguous contigs and reconstructed more reference genes with a combination of multiple contigs, while soaptrans ran much faster than trinity.
these differences in contiguity and completeness became more obvious with the reduced set of input data .
these findings recommend the illumina platform with trinity assembly to obtain the most complete gene coverage by a single contig, especially when a small amount of reads are available.
in instances where a large amount of data is available and there are limited computational resources, illumina soaptrans assembly may be preferred as it generated a relatively complete assembly much more quickly than trinity.
furthermore, evaluation of the amount of illumina sequence data required for generating a complete transcriptome is approximately  <dig> million reads.
geraniaceae organelle genomes have been shown to exhibit a number of unusual features relative to other angiosperms, including highly accelerated rates of nucleotide substitutions in both mitochondrial and plastid genes  <cit> , reduced rna editing in mitochondrial genomes  <cit>  and highly rearranged plastid genomes  <cit> .
this comparative transcriptome analysis of g. maderense and p. x hortorum detected a reduction in ppr proteins associated with rna editing, which corresponds with reduced rna editing in the mitochondria.
examination of nuclear encoded, plastid targeted sigma factors required for pep function supports the hypothesis that pep is active in p. x hortorum plastids, possibly incorporating the product of at least one of the highly divergent rpoa-like orfs in the plastid genome.

 four remarkable evolutionary phenomena are associated with organellar genomes of geraniaceae. first, mitochondrial genomes show multiple, major shifts in rates of synonymous substitutions, especially in the genus pelargonium. rate fluctuations of such magnitude have been documented in only two other plant lineages, plantago and silene. second, mitochondrial genomes have experienced extensive loss of genes and sites of rna editing. at least putative gene losses have been documented in erodium, and mitochondrial genes sequenced from pelargonium x hortorum had a drastic reduction in predicted or verified rna editing sites compared to all other angiosperms examined. third, genome-wide comparisons of nucleotide substitutions in plastid dna indicated rapid rate acceleration in genes encoding ribosomal proteins, rna polymerase, and atp synthase subunits in some lineages. in the case of rna polymerase genes there was evidence for positive selection. fourth, plastid genomes of geraniaceae are the most highly rearranged of any photosynthetic land plants examined. multiple and extreme contractions and expansions of the inverted repeat have resulted in genomes with both the largest ir as well as the complete loss of this feature. considerable accumulation of dispersed repeats associated with changes in gene order has been documented along with disruption of highly conserved operons and repeated losses and duplications of genes. in p. x hortorum plastids, these genomic changes have generated several fragmented and highly divergent rpoa-like orfs of questionable functionality, despite the fact that rpoa encodes an essential component of the plastid-encoded rna polymerase. because nuclear genes supply both organelles with the majority of their proteins, it is likely that the extensive organellar genomic upheaval in geraniaceae will also influence the evolution of organelle-targeted genes in the nuclear genome. for example, given the drastic reduction of rna editing in geraniaceae mitochondrial transcripts, it is reasonable to expect a correlated reduction of nucleus-encoded pentatricopeptide repeat proteins, many of which are critical for organellar rna editing. the uncertain status of the p. x hortorum plastid-encoded rpoa gene is also likely to have nuclear consequences. if this plastid gene is not functional, then a functional copy might have been relocated to the nuclear genome, which has only occurred once in the evolution of land plants in mosses. alternatively, it is possible that pep has become nonfunctional in p. x hortorum, as observed in the holoparasite phelipanche aegyptiaca. in p. aegyptiaca, loss of all plastid-encoded pep components resulted in the parallel loss of the requisite nucleus-encoded components that assemble with the plastid encoded proteins to form the core of the pep holoenzyme. in contrast, if the highly divergent plastid rpoa gene is still functional in p. x hortorum, then the typical set of sigma factors should be present in the nuclear genome. one prerequisite to begin to address the effects of organellar genomic upheaval on the nuclear genome in geraniaceae, is availability of nuclear sequence information. transcriptome sequencing provides a tractable proxy for nuclear gene space. the use of next-generation sequencing for transcriptome sequencing is widespread because volumes of data can be generated rapidly at a low cost relative to traditional sanger sequencing. the assembly of reads into contigs may be executed using a de novo or a reference-based approach. in studies of non-model organisms, de novo assembly is more commonly used due to the absence of a closely related reference. a survey of recent transcriptome studies in comparative biology demonstrates that most sequencing projects are focusing on non-model organisms where little or no genomic data is available. the lack of a reference genome makes the reconstruction and evaluation of the transcriptome assembly challenging. several issues must be addressed when performing transcriptome sequencing of non-model organisms, including which ngs platform should be employed, how much sequence data is needed to provide a comprehensive transcriptome, which assembler should be utilized, and what tissues should be sampled. this paper provides a comprehensive comparison of the transcriptomes of two non-model plant species, pelargonium x hortorum and geranium maderense, from the two largest genera of geraniaceae. there were three primary goals for the initial comparative transcriptome analysis in geraniaceae: what are the best sequencing platforms and assembly methods for generating a high-quality transcriptome that broadly covers gene space in the absence of a reference genome? does sequencing from multiple tissue types improve the breadth of transcriptome coverage? are there any losses of ppr proteins involved in rna editing and sigma factors associated with pep in geraniaceae?  ribosomal rna content and illumina library complexity to assess the efficiency of ribosomal rna depletion in geraniaceae transcriptome libraries rrna contigs were identified using rrna from arabidopsis thaliana as a reference. all illumina reads were mapped to rrna contigs as described in methods, and % and 2% of the reads of g. maderense and p. x hortorum were identified as rrna reads, respectively. library complexity was analyzed using picard and rrna reads were eliminated prior to the analysis. the percentages of unique start sites were % and % for g. maderense and p. x hortorum, respectively. the values for rrna content and library complexity were comparable to other transcriptome analyses using similar approaches. assessment of sequencing platforms and assemblers for transcriptome assembly to determine the optimal sequencing and assembly strategy, the efficacy of five different assemblers was examined using two initial data sets generated by roche/ flx and illumina hiseq platforms for p. x hortorum. the illumina run produced approximately times more sequence data than the run, even though the cost of the data was at least four times more than the illumina data. a comparison of basic assembly statistics showed that the trinity assembler outperformed all other platform/software combinations in terms of number of contigs, number of assembled nucleotides, mean and maximum contig length, and n more generally, the illumina assemblers consistently outperformed the assemblers, although the mira and newbler assemblers produced longer maximal contigs than soapdenovo and soapdenovo-trans. to determine the amount of usable protein sequence information generated by each assembler, the assemblies were translated as described in methods and compared. again, the illumina assemblers outperformed the assemblers in all metrics, with the trinity assembler providing the most amino acids with the longest mean and maximal sequences. the length distribution of assembled nucleotides and translated amino acids further confirms that trinity outperformed soapdenovo and soaptrans, and all three illumina assemblers outperformed the assemblers. pelargonium x hortorum transcriptome dataset read statistics x hortorum transcriptome x hortorum two important considerations in assembly analysis are the breadth of gene space coverage and the degree of coverage fragmentation. a good assembler should generate high-quality assemblies that contain as many reference transcripts as possible, and each reference transcript should be covered as completely as possible with a single long contig rather than a combination of several short contigs. to assess assembly coverage and fragmentation, two published data bases were used, ultra-conserved ortholog coding sequence from arabidopsis and single copy nuclear genes shared between arabidopsis, oryza, populus, and vitis. trinity and soaptrans outperformed all other assemblers in terms of the percentage of reference genes identified, completeness of coverage, and contiguity of coverage, with trinity performance slightly better than soaptrans at higher thresholds. to examine whether the superior performance of trinity and soaptrans was due to the much larger amount of illumina data than data, the illumina assemblers were re-anlayzed using a data set containing 1/40th of the illumina reads. in terms of contiguity and completeness, the performance of trinity using the reduced illumina data set remained superior to the programs that used the entire data sets. in contrast, the performance of soapdenovo and soaptrans were noticeably worse with the reduced illumina data set than with the full data set, producing results that were generally worse than the original assemblies. effect of sequencing depth on assembly coverage breadth and fragmentation to determine how much sequence data is needed to assemble a high-quality transcriptome with broad coverage,, reads for g. maderense and, reads for p. x hortorum were generated on the illumina hiseq platform assembled using trinity with different increments of reads from 5% to 100% of the total. while the number of contigs assembled continued to increase with increasing numbers of reads, the percentage of reference genes recovered and their contiguity and completeness plateaued at approximately 40% of the total reads. including the remaining 60% of the reads increased contiguity and completeness by only 1% to 2%. although there were more translated contigs of g. maderense than p. x hortorum, the contiguity and completeness of both species were very similar. although increasing the number of reads beyond 10% contributed little to finding novel hits to the local arabidopsis data base, increasing the amount of data did help extend the existing contigs and generate longer alignments to reference genes. to evaluate this, the contiguity of all contigs relative to the two published databases was calculated at different contiguity thresholds up to 100%. the inclusion of more reads generated assemblies with higher contiguity, especially when contiguity thresholds were greater than 50%. to allow for the high level of sequence divergence between geraniaceae and arabidopsis, the number of contigs that had contiguity thresholds ≥80% was calculated. when 100% of the reads were used contigs and contigs were found in g. maderense and p. x hortorum, respectively. reducing the read input to 40% reduced contiguity values by 7% in g. maderense and 11% in p. x hortorum. functional assessment of geraniaceae nuclear transcriptomes the assemblies generated using 100% of the reads for both geraniaceae species were used for functional annotation. assemblies were first aligned against the ncbi nr database and the alignment results were used to generate the gene ontology terms. of the contigs in p. x hortorum, had blast hits; were annotated and go terms were retrieved. of the contigs in g. maderense, had blast hits; were annotated and go terms were retrieved. the annotation files are shown in additional file the distribution of gene ontology annotations was examined using go-slim ontology to compare the transcriptomes of g. maderense and p. x hortorum. although the number of annotated contigs differed substantially between the two transcriptomes, the proportion of annotated contigs in all categories with >1% representation within the categories cellular component, molecular function, and biological process were very similar. this similarity persists even though only emergent leaves were sampled for g. maderense versus four tissue types for p. x hortorum. geranium maderense and pelargonium x hortorum go = gene ontology; ec = enzyme code. to more directly address the question whether sequencing from multiple tissue types improves the breadth of transcriptome coverage, orthologous genes between g. maderense and a. thaliana and between p. x hortorum and a. thaliana were identified. of the protein sequences from a. thaliana, the g. maderense assembly had homologs to sequences and the p. x hortorum assembly had homologs to sequences. the comparable numbers of orthologous genes found for the two geraniaceae species indicated that there was little improvement on the breadth of transcriptome coverage by sequencing from multiple tissue types. identification of selected organelle targeted genes pentatricopeptide repeat proteins are a large family of rna binding proteins encoded by over genes in angiosperms; most are organelle targeted and involved in regulating organelle gene expression. the transcriptomes of p. x hortorum and g. maderense were annotated using arabidopsis ppr sequences as a reference database. the overall number of ppr genes varied considerably between the two geraniaceae and arabidopsis, with ppr gene number reduced in p. x hortorum. the numbers of p class ppr genes were found to be similar in all three species, whereas many fewer pls class genes were found in the geraniaceae, especially in p. x hortorum. a and sigma factor b distribution arabidopsis thaliana geranium maderense pelargonium x hortorum appr protein data of arabidopsis are from small and peeters. the ppr class represents the number contigs longer than aa, which is the minimum length of ppr proteins identified in arabidopsis. bthe number of total contigs and the number of intact contigs aligned to the reference sigma factors are shown. intact contigs are those with start/stop codons on 5′ and 3′ ends, and without any internal stop codons. sigma factors are nuclear encoded, plastid targeted proteins that assemble with four plastid encoded proteins to form the core of the pep holoenzyme. at least one copy of each of the six arabidopsis sigma factors was detected in both the g. maderense and p. x hortorum transcriptomes. the nucleotide and amino acid sequence identities between arabidopsis/geranium and arabidopsis/pelargonium for all six sigma factors were very similar. the four contigs from g. maderense that aligned to sigma factor were similar to each other in nucleotide sequence identity, suggesting that they may represent variant copies of the same gene. two of the three contigs from g. maderense that aligned to sigma factor were very similar to each other but less so to the third contig. sigma factors and were each represented by two p. x hortorum contigs, however only one of the contigs for each sigma factor appeared functional having start/stop codons at the 5′ and 3′ ends and lacking internal stop codons. further experiments are needed to determine if the copies with internal stop codons are pseudogenes or assembly artifacts. arabidopsis thaliana sigma factors arabidopsis thaliana geranium maderense pelargonium x hortorum sequence identity a ain cases where there is more than one intact contig for a sigma factor, the one with highest sequence identity to arabidopsis was selected for comparison. discussion strategies for de novo assembly of transcriptomes the use of ngs platforms is widespread and is applied in many research fields as volumes of data can be generated rapidly at a low cost relative to traditional sanger sequencing. rna-seq, one popular ngs application, provides an efficient and cost-effective way of obtaining transcriptome data. there are a number of platforms available for generating ngs data. currently among the most popular are the roche/ flx and the illumina hiseq platforms. the roche/ flx system is advantageous when longer reads are important, whereas the illumina system provides deeper sequencing coverage at a reduced cost per base, albeit with shorter read length. for each platform various assemblers have emerged but during the past several years roche sequencing and the platform-specific assembler newbler has been the most common approach for de novo assembly of transcriptome data. this may be attributed to the idea that longer reads are more likely to overcome the specific challenges of de novo transcriptome assembly. illumina sequencing has been used mainly when a related organism’s genome was available for reference-based assembly, although due to recently increased read length it is becoming more common for use in de novo assembly as well. several recent studies compared the performance of different sequencing platforms and assembly methods but none of these comparisons evaluated the level of completeness or contiguity of their assemblies, nor was the performance of the assemblers evaluated without known genome information, which is the situation for any project on non-model organisms. our comparisons of sequencing platforms and assemblers for the geraniaceae clearly indicated that the illumina platform with trinity assembly delivered the best performance in assembling a complete transcriptome in the absence of a reference genome. the illumina assemblers generated more contigs containing a greater total number of bases than the roche/ flx assemblers. hile the mira assembly generated many more long contigs than soapdenovo, the trinity assembly out-performed all others in delivering long contigs, suggesting that the trinity assembly contained more useful information than any of the other assemblies analyzed. while the roche/ flx assemblies and the illumina soapdenovo assembly produced similar results with regard to completeness and contiguity, the illumina trinity and soaptrans assemblies obtained much higher values for both parameters indicating that these assemblies comprise many more nearly complete transcripts. functional annotation of geraniaceae transcriptomes a total of and contigs were annotated from g. maderense and p. x hortorum, respectively. the low percentage of annotated contigs is most likely due to the large number of total contigs assembled. the number of aligned and annotated contigs is comparable to nine other recently published transcriptomes. the number of annotated contigs in assemblies from both geraniaceae species was very similar for the three major categories cellular component, molecular function, and biological process. this is encouraging since different tissues were sampled for the two species; only one tissue, emergent leaves for geranium and four tissues, emergent leaves, expanded leaves, roots and flowers for pelargonium. particularly noteworthy is the detection of genes associated with flower and embryo development and pollen-pistil interaction since flowers were not sampled for geranium. overall, this comparison indicates that there is no marked improvement in transcriptome breadth of coverage when sampling four tissues compared to only emergent leaves. ppr proteins and sigma factors in geraniaceae pprs are a large family of rna binding proteins encoded by over genes in sequenced angiosperms. most are organelle targeted and involved in regulating organelle gene expression. of the two classes within the ppr family, those from pls class have been reported to be involved in rna editing. previous studies have demonstrated correlated evolution of pls genes and rna editing sites in plants. consistent with these results, a reduction in pls genes in geraniaceae was detected, where reduced editing frequency was previously demonstrated. the reduced editing frequency and reduced ppr content in geraniaceae is especially intriguing with respect to the increased mitochondrial substitution rate in this family. although an inverse correlation between editing frequency and substitution rate has been noted previously in geraniaceae and other taxa, the finding that ppr gene content is also reduced in geraniaceae indicates that this family is ideally suited for future studies assessing the evolutionary dynamics of editing frequency, ppr content, and mitochondrial substitution rates. one long-standing question regarding the plastid genomes in geraniaceae is the putative loss of the rpoa gene from p. x hortorum. the complete plastid genome sequence of this species revealed several rpoa-like open reading frames that are highly divergent relative to rpoa genes in other angiosperms or even other geraniaceae. two alternative explanations were suggested for these observations: a copy of the gene in the nucleus had gained functionality; or at least one of the highly divergent rpoa-like orfs remains functional. extensive evolutionary rate comparisons of plastid genes across the geraniaceae revealed that the other three pep subunits have significantly elevated nucleotide substitution rates and have likely experienced positive selection. despite exhaustive searching of the nuclear transcriptome of p. x hortorum no copy of the rpoa gene was detected. however, intact copies of all six sigma factors, which are required for pep to function, were identified in the transcriptome. the holoparasite phelipanche aegyptiaca lacks a functional pep and mining unigene files published in a recent transcriptomic study of parasitic plants failed to uncover a single sigma factor suggesting that in species where pep sequences are lost from the plastid the requisite sigma factors are also absent from the nuclear transcriptome. the identification of all six sigma factors in the p. x hortorum transcriptome supports the likelihood that pep is active in p. x hortorum plastids.  with the widespread application of ngs techniques, the ability to process and analyze massive quantities of sequence data in a timely manner becomes imperative to a successful project. regardless of the goals of a particular project, it is desirable to obtain data that is as accurate and complete as possible in a way that is cost effective as well as timely. in this study a cross-platform comparison of de novo transcriptome assembly was conducted using representative species from the two largest genera of geraniaceae, g. maderense and p. x hortorum. as no reference genome is available for geraniaceae, or any of its close relatives, this approach represents a truly de novo assembly allowing evaluation of efficacy among the platforms/assemblers that more closely resembles current ngs research. the assembly of illumina hiseq reads with trinity or soaptrans was highly effective in reconstructing, as completely as is currently feasible, the protein-coding transcripts of geraniaceae. as for the differences between the two assemblers, trinity generated slightly more single contiguous contigs and reconstructed more reference genes with a combination of multiple contigs, while soaptrans ran much faster than trinity. these differences in contiguity and completeness became more obvious with the reduced set of input data. these findings recommend the illumina platform with trinity assembly to obtain the most complete gene coverage by a single contig, especially when a small amount of reads are available. in instances where a large amount of data is available and there are limited computational resources, illumina soaptrans assembly may be preferred as it generated a relatively complete assembly much more quickly than trinity. furthermore, evaluation of the amount of illumina sequence data required for generating a complete transcriptome is approximately million reads. geraniaceae organelle genomes have been shown to exhibit a number of unusual features relative to other angiosperms, including highly accelerated rates of nucleotide substitutions in both mitochondrial and plastid genes, reduced rna editing in mitochondrial genomes and highly rearranged plastid genomes. this comparative transcriptome analysis of g. maderense and p. x hortorum detected a reduction in ppr proteins associated with rna editing, which corresponds with reduced rna editing in the mitochondria. examination of nuclear encoded, plastid targeted sigma factors required for pep function supports the hypothesis that pep is active in p. x hortorum plastids, possibly incorporating the product of at least one of the highly divergent rpoa-like orfs in the plastid genome. 
an analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million illumina paired end reads for both species. the findings support the use of the illumina platform and assemblers optimized for transcriptome assembly, such as trinity or soaptrans, to generate high-quality de novo transcriptomes with broad coverage. since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. to begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, geranium maderense and pelargonium x hortorum. trinity provided the highest quality assembly of illumina data with the deepest transcriptome coverage. in addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling rna from four tissue types rather than from a single tissue. organelle genomes of geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced rna editing, and extensive genomic rearrangements. the g. maderense and p. x hortorum transcriptomes contained fewer transcripts encoding the pls subclass of ppr proteins relative to other angiosperms, consistent with reduced mitochondrial rna editing activity in geraniaceae. normalized cdna libraries of g. maderense and p. x hortorum were used for transcriptome sequencing. finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants. in addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoa-like orfs in the p. x hortorum plastid genome is functional.
 organelle genomes of geraniaceae exhibit several unusual evolutionary phenomena compared to other angiosperm families including accelerated nucleotide substitution rates, widespread gene loss, reduced rna editing, and extensive genomic rearrangements. since most organelle-encoded proteins function in multi-subunit complexes that also contain nuclear-encoded proteins, it is likely that the atypical organellar phenomena affect the evolution of nuclear genes encoding organellar proteins. to begin to unravel the complex co-evolutionary interplay between organellar and nuclear genomes in this family, we sequenced nuclear transcriptomes of two species, geranium maderense and pelargonium x hortorum.  normalized cdna libraries of g. maderense and p. x hortorum were used for transcriptome sequencing. five assemblers and two next-generation technologies were compared to determine the optimal transcriptome sequencing approach. trinity provided the highest quality assembly of illumina data with the deepest transcriptome coverage. an analysis to determine the amount of sequencing needed for de novo assembly revealed diminishing returns of coverage and quality with data sets larger than sixty million illumina paired end reads for both species. the g. maderense and p. x hortorum transcriptomes contained fewer transcripts encoding the pls subclass of ppr proteins relative to other angiosperms, consistent with reduced mitochondrial rna editing activity in geraniaceae. in addition, transcripts for all six plastid targeted sigma factors were identified in both transcriptomes, suggesting that one of the highly divergent rpoa-like orfs in the p. x hortorum plastid genome is functional.  the findings support the use of the illumina platform and assemblers optimized for transcriptome assembly, such as trinity or soaptrans, to generate high-quality de novo transcriptomes with broad coverage. in addition, results indicated no major improvements in breadth of coverage with data sets larger than six billion nucleotides or when sampling rna from four tissue types rather than from a single tissue. finally, this work demonstrates the power of cross-compartmental genomic analyses to deepen our understanding of the correlated evolution of the nuclear, plastid, and mitochondrial genomes in plants. 