text:
Mutation spectrum of Drosophila CNVs revealed by breakpoint sequencing
- Margarida Cardoso-Moreira1Email author,
- J Roman Arguello1 and
- Andrew G Clark1
DOI: 10.1186/gb-2012-13-12-r119
© Cardoso-Moreira et al.; licensee BioMed Central Ltd. 2013
Received: 31 May 2012
Accepted: 22 December 2012
Published: 22 December 2012
The detailed study of breakpoints associated with copy number variants (CNVs) can elucidate the mutational mechanisms that generate them and the comparison of breakpoints across species can highlight differences in genomic architecture that may lead to lineage-specific differences in patterns of CNVs. Here, we provide a detailed analysis of Drosophila CNV breakpoints and contrast it with similar analyses recently carried out for the human genome.
By applying split-read methods to a total of 10x coverage of 454 shotgun sequence across nine lines of D. melanogaster and by re-examining a previously published dataset of CNVs detected using tiling arrays, we identified the precise breakpoints of more than 600 insertions, deletions, and duplications. Contrasting these CNVs with those found in humans showed that in both taxa CNV breakpoints fall into three classes: blunt breakpoints; simple breakpoints associated with microhomology; and breakpoints with additional nucleotides inserted/deleted and no microhomology. In both taxa CNV breakpoints are enriched with non-B DNA sequence structures, which may impair DNA replication and/or repair. However, in contrast to human genomes, non-allelic homologous-recombination (NAHR) plays a negligible role in CNV formation in Drosophila. In flies, non-homologous repair mechanisms are responsible for simple, recurrent, and complex CNVs, including insertions of de novo sequence as large as 60 bp.
Humans and Drosophila differ considerably in the importance of homology-based mechanisms for the formation of CNVs, likely as a consequence of the differences in the abundance and distribution of both segmental duplications and transposable elements between the two genomes.
KeywordsCopy number variants CNVs Non-allelic homologous-recombination NAHR Single-strand annealing SSA Non-homologous end-joining NHEJ Replication-associated repair Alternative end-joining Microhomology-mediated end-joining MMEJ Filler DNA
One of the most surprising discoveries about genome sequence variation was the finding that copy number variants (CNVs; that is, duplications, deletions, and insertions) are widespread in eukaryotic genomes. CNVs have the potential to create novel genes, to alter gene structures, and/or to change gene regulation. As a result, CNVs can cause large phenotypic effects, ranging from highly deleterious [1, 2], to CNVs underlying adaptation to novel environments [3, 4]. The phenotypic effects of CNVs shape their genomic distribution: in natural populations, CNVs are strongly depleted among protein-coding genes and other functional elements of the genome [5, 6]. However, in addition to selection, mutational processes also impact the genomic distribution of CNVs [7–10]. The distribution of these variants is not uniform across the genome; instead, CNVs accumulate in discrete regions as a consequence of local increases in the mutation rate. Consequently, current efforts aimed at the identification of the causal CNVs of both deleterious and adaptive phenotypes could be greatly enhanced by a better understanding of the mutational processes underlying the formation of CNVs and the genomic features associated with elevated mutation rates.
CNVs are formed when the repair of DNA breaks (mostly DNA double-strand breaks) is not perfect, leading to the creation of copy-number mutations. DNA double-strand breaks arise as part of the normal metabolism of the cell or as a consequence of ionizing radiation or reactive oxygen species [11, 12]. There are three molecular pathways available to repair the breaks, two that require sequence homology to perform the repair - homologous recombination (HR) and single-strand annealing (SSA) - and one that is homology-independent - non-homologous end-joining (NHEJ). Although both HR and SSA require sequence homology to repair DNA double-strand breaks, they differ in the extent of homology that is required: 100 to 200 bp for HR versus as little as 50 bp for SSA [11, 12]. Another difference is that while SSA always creates a deletion as a consequence of the repair (it is a mutagenic repair pathway), most of the time HR repairs the DNA break without generating any mutation. However, the existence of segmental duplications (also called low copy repeats (LCRs)) or transposable elements near the DNA break can lead to misalignments in the region. In this case, the repair occurs between misaligned repeats leading to the formation of duplications and deletions in a process known as non-allelic homologous recombination (NAHR). In the absence of sequence homology the cell can use non-homologous pathways to repair DNA double-strand breaks. NHEJ, like SSA, is mutagenic, usually resulting in nucleotide substitutions or small indels, but it can also create larger insertions and deletions. While NHEJ does not require sequence homology, a related alternative end-joining pathway, microhomology-mediated end-joining (MMEJ), uses microhomology to mediate the repair [13]. The different molecular pathways are therefore associated with different types of breakpoints and classes of CNVs: NAHR is associated with large stretches of sequence identity and generates both duplications and deletions; SSA is associated with smaller stretches of sequence identity and only generates deletions; NHEJ and its associated pathways (for example, MMEJ) are associated with either presence (2 to 10 bp) or absence of microhomology, and are mostly associated with deletions and insertions (although it can also generate duplications) [11, 12].
In recent years, additional molecular mechanisms have been proposed to operate in association with replication-based repair and cause CNVs. These mechanisms were proposed following the observation that a subset of human CNVs are highly complex [12, 14, 15]. Such complex CNVs are hard to explain given the canonical HR (and the associated NAHR) and NHEJ pathways because they would require multiple DNA double-strand breaks. Furthermore, the analysis of the breakpoints of these CNVs suggested multiple rounds of strand invasion and the copying of nearby sequences [12, 14, 15], signatures that could more easily be explained by replication forks stalling (or collapsing), and subsequently disengaging from the template and re-annealing. Three of the proposed models are: fork stalling and replication switching (FoSTeS) [16], microhomology-mediated break-induced replication (MMBIR) [17], and serial replication slippage (SRS) [18]. Although these models differ in specific details [12, 14], they are essentially indistinguishable in terms of breakpoint analysis. They all share the requirement that the re-annealing is mediated by microhomology, and they also suggest that templated DNA from nearby sequences can be introduced at the breakpoints [12, 14]. Although these models have also been proposed to mediate the formation of simple CNVs, it is challenging to distinguish the signatures of these microhomology-mediated replication models from those of NHEJ (and associated MMEJ). In principle, one could distinguish between the two when there are additional nucleotides present at CNV breakpoints: replication-based models would predict that the additional nucleotides correspond to templated DNA (that is, the extra nucleotides were copied from a nearby location) while NHEJ/MMEJ would predict that the additional nucleotides correspond to filler DNA (that is, the extra nucleotides were randomly incorporated).
Most of the work in CNV breakpoint identification has been restricted to mammalian genomes, and in particular to the human genome [19–23]. In humans (as in other mammals) CNVs are significantly enriched close to segmental duplications [8, 12]. These regions were initially proposed, and subsequently shown to be, CNV hotspots predominantly through facilitating NAHR [7, 8, 12]. However, not all human CNV hotspots are associated with segmental duplications; in fact, a sizeable fraction is not [7, 21]. Here, we aim to further our understanding of the mutational mechanisms underlying the formation of CNVs by extending breakpoint analysis to the D. melanogaster genome. CNVs are as widespread in the fly as in mammalian genomes [5, 24, 25], and CNV hotspots have been identified in both D. melanogaster [9] and its sister species, D. simulans [10]. Although patterns of copy number variation share many similarities between humans and flies, the two genomes have very different genomic architectures. For example, while segmental duplications comprise approximately 5 % of the human and mouse genomes, they comprise only 1% of the fly genome [26]. Similarly, while transposable elements comprise approximately 50% of the human genome, they only correspond to 20% of the fly genome, where they are mostly restricted to pericentromeric regions and the fourth chromosome [27]. (The same holds true for segmental duplications [26].) Our goal was to take advantage of the differences in genome architecture between flies and humans in order to dissect the contribution of different genomic features to the formation of CNVs. We have done this by examining two distinct sets of CNVs: one generated using long Roche/454 sequencing reads [28] and the other using high-resolution tiling microarrays [5]. The use of these two dataset sets has enabled us to overcome many of the potential biases associated with each individual method if used alone. Our results indicate that fly CNVs share several of the striking characteristics observed for human CNVs: (1) a paucity of breakpoints associated with both microhomology and additional nucleotides inserted/deleted at the breakpoints; (2) an enrichment of non-B DNA sequences at the CNV breakpoints; and (3) a significant fraction of both recurrent and complex CNVs. Importantly, however, the different architectural organization of the fly genome does appear to shape patterns of copy number variation: homology-based pathways (notably NAHR) play a minor role in the formation of fly CNVs, including recurrent CNVs. Our data indicate that in flies non-homologous pathways underlie most CNV formation for both simple and complex events. One important consequence is that in flies most insertions do not correspond to duplications of previously existing sequence but are instead created de novo by the random insertion of nucleotides and/or small repeats from nearby sequences.
Precise breakpoint detection of CNVs from a 454 sequencing dataset
Sackton and colleagues sequenced at low coverage (approximately 0.2x) the genomes of nine D. melanogaster strains using Roche/454 technology [28]. These genome sequences were used to evaluate the extent to which population genomic inferences could be made from low/sparse genomic coverage. Sackton and colleagues identified not only SNPs, but also transposable elements and CNVs. However, the latter were identified using a paired-end framework that did not provide the exact breakpoints of the CNVs. Here, we employ a different approach to detect CNVs based on split-read mapping that is capable of detecting CNVs with precise breakpoint resolution (that is, single nucleotide resolution). Defining what is the minimum size of a variant for it to be considered a CNV as opposed to an indel is largely arbitrary and often reflects the degree of resolution of the platform used to identify those variants. While initial CNV studies defined these variants as being at least 1 kb in length, more recent studies (for example, 1000 Genomes Project [21]) use 50 bp as the lower limit for calling a variant a CNV. In agreement with the previous literature on Drosophila CNVs [5, 10], here we use 25 bp as the lower limit to classify insertions, deletions, and duplications as CNVs.
We downloaded the raw data for the nine genomes sequenced by Sackton and colleagues [28] and aligned the reads against the D. melanogaster reference genome using the aligner Mosaik [29]. We discarded all reads that mapped to the reference genome and focused only on the subset of the reads that failed to map. We re-aligned these reads to the reference genome using BLAT [30] (see Methods). Because BLAT was designed to align mRNA onto genomic DNA, it does not penalize the existence of large gaps between the reads and the reference genome and provides the exact location of those gaps. By parsing the BLAT results we identified all reads that: (1) had a deletion larger than 25 bp in relation to the reference; (2) had an insertion larger than 25 bp in relation to the reference; and (3) mapped to two different locations with the 3' end of the read mapping 5' of the 5' end of the read (the pattern created by a tandem duplication). Because the nine genomes were sequenced at low coverage, our goal was not to identify all existing CNVs but instead to create a high-quality dataset of CNV breakpoints. To that effect, we applied a series of filters to minimize false-positive calls. Briefly, we required that each breakpoint was seen in at least two independent reads (from the same genome or from different genomes), that those two reads were not PCR duplicates, that the breakpoint was not located within the last 10 bp of the ends of the reads and that the breakpoint mapped to the euchromatic region of the genome. We also excluded from the dataset all deletions/insertions that corresponded to transposable element polymorphisms (that is, the deleted/inserted sequence mapped exclusively to annotated transposable elements). Finally, we identified the exact breakpoint configuration by re-aligning the reads supporting each of the breakpoints to the reference genome sequence using Clustal [31, 32].
Using this pipeline, we identified 447 deletions and 197 insertions larger than 25 bp segregating in the nine genomes. Because we required that at least two independent reads supported each breakpoint we biased our sample toward CNVs segregating in multiple genomes (as opposed to being private to one of the genomes). A total of 72% of CNV calls are supported by reads from at least two of the nine genomes, with only 28% of the CNVs supported by multiple reads from the same genome. This result is expected given the sparseness of the genomic data.
We evaluated the quality of our calls by confirming a subset of these variants by PCR and Sanger sequencing. Out of 32 CNVs tested, all were confirmed by PCR and sequencing. Sanger sequencing supported not only the existence of the CNVs but also the precise breakpoint configuration. We tested an additional set of eight CNVs that were filtered out from the final dataset because the reads supporting them were potential PCR duplicates. Again, all eight CNVs were confirmed, suggesting this was a fairly conservative filter. However, because our pipeline was able to identify a large number of CNV breakpoints (n = 644), and because our focus is on inference of mechanisms of CNV formation from sequence patterns using high-confidence CNV calls, we favored the more conservative dataset that minimized the number of false-positives.
To investigate the existence of potential differences between the mutational mechanisms underlying the formation of insertions and deletions, we used the D. simulans reference genome, and a parsimony approach, to polarize the calls (see Methods). Out of 447 deletions, 338 were confirmed to be deletions segregating in the sequenced D. melanogaster strains, 13 were re-classified as insertions in the reference genome, and 96 could not be polarized. Out of 197 insertions, 37 were confirmed to be insertions segregating in the sequenced D. melanogaster strains, with 123 being re-classified as deletions in the reference genome, and 37 could not be polarized.
Most insertions are not tandem duplications and correspond to de novo DNA
After polarization, our dataset included 50 insertions: 13 present in the reference genome sequence and 37 segregating in the strains sequenced. Of the 50 insertions, only two (4%) are tandem duplications, whereby the inserted sequence is a copy of a stretch of DNA already present in the genome (at a nearby location). Of the remaining 48 insertions, seven (14%) correspond to simple expansions of dinucleotides or small repeats flanking the insertions, and 41 (82%) have no match to the reference genome sequence and were thus classified as 'filler DNA' [13]. Filler DNA is a common outcome of the repair of DNA double-strand breaks by NHEJ in flies [33, 34] and other organisms [35]. Filler DNA has been observed in several studies of DNA repair that use artificial DNA constructs where DNA double-strand breaks are induced and the products of the DNA repair can be recovered and sequenced. In most cases, only a few nucleotides (or none) are added to the repaired junctions, but in some instances large insertions are created [13, 33, 35].
These data suggest that most insertions in D. melanogaster do not correspond to tandem duplications or to expansions of di- or tri-nucleotides or repeats, but instead that they are the product of the random incorporation of nucleotides and of the copying of small stretches of DNA from nearby sequences as part of the process of DNA repair. Although anecdotal, the fact that the two tandem duplications identified are also two of the largest insertions might be interpreted as suggesting that larger insertions may indeed correspond mostly to tandem duplications while smaller insertions (that is, smaller than 60 bp) will mostly correspond to novel stretches of DNA sequence. The observation that most insertions in Drosophila correspond to novel DNA sequence contrasts with a previous observation made for the human genome, where most recent insertions (1 to 100 bp; appeared after the human-chimpanzee split) were determined to correspond to tandem duplications [36].
Distinct classes of CNV breakpoints
Comparison of the types of CNV breakpoints identified in Drosophila and humans.
Type of breakpoint
Human (Conrad et al.)a
Human (Kidd et al.)b
Molecular mechanism(s)
MMEJ, replication-associated repair
Blunt and large stretches of sequence identity (³20 bp)
SSA, NAHR, replication-associated repair
Inserted/deleted bases
NHEJ, replication-associated repair
Inserted/deleted bases and microhomology
MMEJ, replication-associated repair
The definition of what constitutes a breakpoint associated with microhomology differs across studies with some authors requiring only 1 bp of identical sequence at the breakpoint (for example, [19]), while others require a minimum of 2 bp or more (for example, [20]). In order to determine the minimum number of identical nucleotides present at a breakpoint that are functionally relevant for CNV formation, we determined the number of nucleotides associated with three distinct types of microhomology for each of the 644 breakpoints in our dataset (Additional file 1, Figure S1). Microhomology of type I is the mechanistically-relevant form of microhomology associated with CNV formation: the deletion occurs between two sequences with microhomology such that one of the sequences becomes part of the deletion (the converse occurs in the case of an insertion). Microhomologies of types II and III (Additional file 1, Figure S1A) are not mechanistically associated with the formation of CNVs but can be used to determine the empirical expectation of finding a similar sequence of n nucleotides close to the breakpoints by chance. As shown in Additional file 1, Figure S1B, only for 2 bp or more do we find a significant excess of microhomology of type I versus the other two types (proportions test, P = 2.2 × 10-12). As a result, in this study we required a minimum of 2 bp of identical sequence to classify a breakpoint as showing evidence for microhomology. Of the 248 deletions associated with microhomology, only two have a stretch of microhomology >20 bp. Thus, at most only two of the deletions in our dataset could have been created by SSA. This is likely to be an over-estimate because previous work in Drosophila has suggested that larger stretches of sequence identity are required to mediate SSA [37]. All other CNVs associated with microhomology consequently are either the product of NHEJ, MMEJ, or of replication-associated repair.
CNV breakpoints harboring complex ends (that is, additional bases present at the breakpoint) are significantly larger than CNVs associated with blunt ends, irrespective of the presence/absence of microhomology (median size of 43 bp vs. 32 bp, Wilcoxon rank sum test P = 1 × 10-13). For 10 of 157 breakpoints with complex ends, the stretches of additional nucleotides inserted are large enough (>20 bp) that they could potentially be mapped to the genome. If replication-based repair mechanisms are involved, the sequences of inserted bases are expected to map to the genome, often close to the deleted sequences. If NHEJ (or a form of alternative end-joining) is involved, the inserted bases should correspond to randomly inserted nucleotides and/or to rearrangements of repeats from nearby sequences (as seen for most insertions). There is no good genomic sequence match for any of the stretches of inserted bases. Furthermore, for seven of the 10 breakpoints, there are small stretches of identity between the inserted bases and nearby sequences that resemble the type of alignments seen between de novo DNA insertions and nearby sequences. These data favor the hypothesis that these CNVs are a consequence of NHEJ or alternative end-joining repair.
Table 1 compares the types of breakpoints identified in this study with those of two previous surveys of human CNV breakpoints [19, 20]. There are two main differences between the types of breakpoints observed in Drosophila and in humans. The first is that in Drosophila there is a higher proportion of blunt breakpoints, a common outcome of NHEJ (35% in Drosophila vs. 11% to 19% in humans). The second is that in Drosophila breakpoints are rarely associated with large stretches of high sequence identity, the hallmark of SSA and NAHR, while in humans Kidd and colleagues found that almost one-third of all breakpoints bore the hallmarks of these pathways [20]. As is clear from Table 1, the two surveys of human CNVs found a very different proportion of breakpoints potentially associated with NAHR (1% in Conrad et al. vs. 29% in Kidd et al.). This difference is likely a consequence of the different experimental approaches used between the studies. Conrad and colleagues used a microarray capture strategy to identify the breakpoints of a subset of CNVs identified in a previous study [6], which may have biased their sample against CNVs associated with NAHR. Kidd and colleagues, on the other hand, identified CNV breakpoints using capillary sequencing of fosmid clone inserts, a powerful approach to sample the full spectrum of CNVs. Further support for a sizeable portion of human CNVs being associated with NAHR, comes from two other studies: one estimated that approximately 28% of breakpoints are associated with NAHR [22] and the other put it closer to 10% to 15% [6]. Motivated by the observation that different technological approaches can produce different results regarding the role played by NAHR in the formation of CNVs, we decided to re-analyze a dataset of 3,639 Drosophila CNVs identified using high-resolution tiling arrays [5] and determine if the observation that there is a paucity of Drosophila CNVs associated with NAHR is robust to the CNV detection platform used.
NAHR plays a minor role in the formation of CNVs in Drosophila
Emerson and colleagues [5] used tiling arrays covering the Drosophila genome at a resolution of 36 bp to identify 3,639 CNVs (2,211 duplications and 1,428 deletions) segregating in the genomes of 15 worldwide strains of D. melanogaster. Microarrays can only probe unique regions of the genome (that is, the probes in the microarray have to map to a unique genomic location), which means that they are biased against detecting additional duplications of regions of the genome that have already been recently duplicated. However, they are unbiased at detecting duplications of unique sequence where copy number changes from one copy (two copies in a diploid genome) to two copies (three or four copies in a diploid genome depending on the duplication being homozygous or heterozygous), irrespective of the presence/absence of flanking duplications. Therefore, we examined the breakpoints of these 3,639 CNVs for the presence of stretches of high-sequence identity in order to determine the contribution of homology-based mechanisms (such as SSA and NAHR) to the formation of CNVs in Drosophila.
Unlike the 454 data, microarray data do not provide the exact breakpoint location. As a consequence, to look for the presence of stretches of high-sequence identity we considered the sequences 500 bp upstream and downstream the predicted CNV breakpoint and the CNV sequence itself. We looked for two types of sequence homology: (1) stretches at least 30 bp in size with a sequence identity of at least 98% (type I; hallmark of SSA); and (2) stretches at least 200 bp in size with a sequence identity of at least 95% (type II; hallmark of both NAHR and SSA) (see Methods).
We found that only 2% (74/3639) of all CNVs were associated with sequence homology of type I (capable of mediating SSA), and 2.6% (95/3639) with sequence homology of type II (capable of mediating SSA or NAHR). Because deletions in this dataset were associated with a high false-positive rate (47%), we also restricted these analyses only to duplications (false-positive rate of 14%). Among the set of duplications, only 2.1% (46/2211) are associated with sequence homology type I, and 2.3% (51/2211) with sequence homology of type II. Therefore, these results support the observation made using the 454 reads that homology-based mechanisms (SSA and NAHR) play a very limited role in the formation of CNVs in Drosophila.
Because both next generation sequencing and microarray technologies are biased against the detection of CNVs in non-unique regions of the genome (that is, segmental duplications and transposable elements) inferences about the importance of homology-based mechanisms are necessarily restricted to unique regions of the genome. However, unlike the human genome where segmental duplications and transposable elements can be found throughout the euchromatin, in Drosophila most repetitive elements are confined to the regions surrounding the centromeres (which have very low rates of recombination) with only a minority of these elements present in regions of the euchromatin with normal recombination rates [26, 27]. Hence, our work suggests that outside of pericentromeric and telomeric regions, homology-based mechanisms play a minor role in CNV formation in Drosophila.
Very high rate of CNV recurrence in Drosophila
CNVs are classified as recurrent when different individuals carry independent but overlapping CNVs. The proportion of recurrent CNVs in the human genome has been estimated to be between 6% and 29% [6]. The sparseness of the 454 dataset prevents us from estimating from these data the proportion of recurrent CNVs in Drosophila. Therefore, in order to evaluate whether CNV recurrence is a common phenomenon in this taxon, we selected 26 genomic regions known to harbor at least one deletion in at least two strains based on the high-resolution tiling array dataset, and screened them in 15 worldwide strains by PCR and Sanger sequencing. These deletions are all located in the euchromatin, their mean size is similar to the mean size of the whole set of deletions and were predicted to range in frequency from 2 to 11 (median 2). Among the 26 regions, 12 (46%) harbored more than one overlapping CNV, suggesting a high rate of CNV recurrence in Drosophila.
Sanger sequencing of these 26 regions showed that the CNVs identified with the tiling arrays have identical characteristics to those identified with the 454 reads. There is no difference in the distribution of breakpoints types present in the 454 dataset and in the set of 36 CNVs (33 deletions and three insertions) segregating in the 26 regions described above (a total of 42 CNVs were detected but for six (mostly tandem duplications) the breakpoints were not fully sequenced). In addition, similar to what was observed in the 454 dataset, CNVs with breakpoints harboring additional bases were, on average, larger than CNVs with simple breakpoints (that is, blunt ends with or without microhomology) (median 432 bp vs. 211 bp, respectively; Wilcoxon rank sum test, P = 0.005).
There was no difference in the distribution of breakpoint types between recurrent and non-recurrent CNVs. Furthermore, just as seen for the non-recurrent set, the recurrent CNVs were not associated with large stretches of sequence identity that might suggest their generation through NAHR. Instead, these data suggest that recurrent CNVs are mediated by non-homologous repair mechanisms. Among the 12 regions showing recurrent CNVs, three also show evidence for the presence of complex CNVs. These occur when a single mutational event generates several breakpoints, that is multiple closely located CNVs segregating within the same individual. In these three regions the distance between distinct breakpoints ranged from 82 bp to 325 bp. This association between complex CNVs (multiple CNVs within the same individual) and recurrent CNVs (multiple CNVs segregating in different individuals) suggests that some regions of the Drosophila genome are particularly unstable, and generate both complex events within individuals as well as independent but overlapping mutations between individuals. Though the sample size is small, these data suggest that complex CNVs may correspond to as much as 12% (3/26) of all Drosophila CNVs, a higher proportion than the 5% estimated for the human genome [6].
Non-B DNA structures are enriched in CNV breakpoints
DNA conformations that do not correspond to the right-handed Watson-Crick double-helix are collectively termed non-B DNA [38, 39]. These include sequences with Z-DNA motifs, quadruplex-forming motifs, inverted repeats, mirror repeats, and direct repeats [40]. Non-B DNA sequences have been found associated with the causal variants of several human diseases and have been proposed to cause genetic instability by impairing DNA repair and DNA replication [38, 39]. Because errors during DNA repair and DNA replication are the ultimate causes of CNVs, we tested for the presence of non-B DNA sequence at the CNV breakpoints identified using the 454 data.
Some classes of non-B DNA sequences are more common than others (in both CNVs and control sequences), but for most we found a shift in the location of these repeats/motifs towards the CNV breakpoint when compared to the control sequences (Additional file 2, Figure S2), suggesting that most classes of non-B DNA sequences are associated with CNV formation. We found the non-B DNA sequences equally associated with the three classes of breakpoints (that is blunt breakpoints, breakpoints associated with microhomology, and breakpoints containing additional nucleotides inserted or deleted; Fisher's exact test, P = 0.98). However, we found a significantly higher proportion of insertions associated with non-B DNA sequences than deletions (Fisher's exact test, P = 0.002). The presence of non-B DNA sequences at a significant fraction of CNV breakpoints suggests a potential causal role for these sequences in CNV formation in flies.
The detailed analysis of Drosophila CNV breakpoints suggests that non-homologous repair mechanisms are responsible for the formation of the majority of the variants. This is true not only for simple CNVs, but also for those that are recurrent and complex. We excluded a significant role for homology-based pathways (that is, NAHR and SSA) in the formation of CNVs because only a minority of these variants (approximately 3%) are flanked by stretches of high sequence identity. We also found little support for replication-associated mechanisms; the large stretches of additional nucleotides present at 10 breakpoints consisted of filler DNA, a result more consistent with NHEJ than with replication-associated repair. The presence of microhomology at CNV breakpoints is, however, consistent with NHEJ, MMEJ, and replication-associated repair (for example, [41]). Determining exactly which pathway(s) are responsible for the different types of CNV breakpoints identified in our study will require the analysis of CNV breakpoints from fly mutants lacking the specific genetic requirements for each pathway (for example, [42, 43]).
In the human and mouse genomes, NHEJ/MMEJ also underlie a large fraction of CNVs, though a sizeable fraction of CNVs are also mediated by NAHR (approximately 18% to 35%) [19–22, 44]. The difference in the preponderance of NHEJ/MMEJ in flies and mammalian genomes does not have to reflect intrinsic differences between these taxa in the relative usage of the different repair pathways (HR vs. NHEJ). In fact, NAHR and SSA are highly efficient in repairing DNA double-strand breaks in flies when these occur in artificial constructs flanked by repeats that can mediate these pathways [37]. Instead, the difference we observe between the taxa in the preponderance of homology-based mechanisms to the formation of CNVs likely reflects the different genomic architectures of the genomes: abundant and widespread presence of segmental duplications and transposable elements throughout mammalian genomes and less abundant and more restricted location (to pericentromeric and telomeric regions) of these elements in the Drosophila genome.
The Drosophila CNVs used in this work are significantly smaller than the published human CNVs. As a consequence, there is the possibility that some of the differences found between the two taxa may reflect different mutational mechanisms operating on CNVs of different size. We note, however, that in flies we found no differences between the breakpoints identified using the 454 data and those found using the high-resolution tiling array data despite the fact that the latter are significantly larger. Although the CNVs identified with the high-resolution tiling arrays are still smaller than those identified for the human genome, their size range already shows a significant overlap with that of the human CNVs used in this study. The absence of large CNVs in the fly genome likely reflects the much higher compactness of this genome (a much higher gene density means that large CNVs would overlap multiple genes) and the greater strength of purifying selection.
We have attempted to circumvent technical biases in CNV detection by making sure that our observations were robust to the platforms used to identify CNVs (that is, both next generation sequencing and hybridization based platforms). Still, our conclusions have to be necessarily restricted to the euchromatic sequence located outside of both pericentromeric and telomeric regions. The latter are highly enriched with transposable elements and segmental duplications making CNV detection extremely challenging irrespective of the platform used. The analysis of the CNV data generated by tiling arrays suggests both a high level of CNV recurrence and complexity (and coupling between the two) that can only be fully explored with technologies that are not biased against the detection of these classes of variants. The development of strobe sequencing technology and of ever larger reads [44] will greatly enhance this effort. By collecting a large number of breakpoints from a sparse low-coverage genomic dataset, we have demonstrated that the analysis of CNV breakpoints does not depend on high coverage datasets, instead read size is likely to matter the most.
Despite a minor role for NAHR, the fly genome is still punctuated by CNV hotspots [9, 10], that is, regions of the genome experiencing higher CNV mutation rates. These hotspots may share the properties of the mammalian CNV hotspots that are not associated with NAHR [7, 45]. Unlike NAHR-mediated hotspots, where the reason for genomic instability is relatively well understood (that is, the presence of repeats leads to misalignments between homologous regions during DNA repair leading to further duplications/deletions), it is not known what causes the instability associated with the remaining hotspots. One possibility is that these correspond to regions more prone to DNA breaks and/or that are harder to repair. In support of this hypothesis we do find that non-B DNA sequences, which are capable of impairing both DNA replication and DNA repair, are significantly enriched at CNV breakpoints.
One of the most surprising results stemming from this work is that in Drosophila most insertions correspond to de novo sequences. These novel stretches of DNA sequence are as large as 60 bp in our dataset, potentially translating into the addition of 20 novel amino acids to a protein sequence. While it is likely that most of these insertions are deleterious, the occasional beneficial mutation could dramatically change the protein sequence in one single mutational event. This would allow very fast protein sequence evolution between closely related species. The frequent creation of novel stretches of DNA sequence as a consequence of DNA repair could have implications for the generation of genetic novelties and genome evolution in general.
Our results suggest that the different architectural features of the Drosophila and human genomes shape the mutation processes responsible for generating duplications, deletions, and insertions. Homology-based pathways contribute significantly more to the formation of CNVs in humans than Drosophila because of the abundance and widespread presence of segmental duplications and transposable elements in humans that can mediate HR. Instead, non-homologous repair is responsible for most CNVs in flies, including complex and recurrent CNVs. Non-homologous repair is also responsible for the creation of insertions made of de novo sequence, which have the potential to mediate rapid protein evolution. In addition, we show that non-B DNA sequences are enriched at CNV breakpoints, which makes these sequences good candidates for being associated with regions of higher CNV instability.
Detection of CNV breakpoints in the 454 data
Split-read methods were first applied to long Sanger sequencing reads [45] and CNVs were detected by identifying those reads which, when mapped to the reference genome exhibit a 'split' signature, either a gap in the reference genome (which suggests an insertion in the read, Additional file 3, Figure S3), a gap in the read (which suggests a deletion in the read, Additional file 3, Figure S3), or two sections of the read mapping to the genome with their positions flipped (which suggests a tandem duplication). Until recently, split-read methods were not widely used because of the small size of the reads produced by next generation sequencing platforms. Roche/454 technology is capable of generating reads >100 bp in size, however eukaryotic genome sequencing projects have predominantly relied on Illumina reads, which only recently achieved the 100 bp mark [46]. With these longer reads, split-read methods can readily identify with precise resolution the breakpoints of duplications, deletions, insertions, inversions, and translocations, as recently shown by the 1000 genomes project [21].
Sackton and colleagues used 454 technology to sequence at low coverage (approximately 0.2x) the genomes of nine D. melanogaster strains (three from an African population and six from a North Carolina population) [28]. We downloaded the original 454 reads (mean read size of 105 bp) from the Short Read Archive (SRP001156) and aligned them to the release 5 of the D. melanogaster genome using Mosaik (version 1.1.0021) [29]. We used the following Mosaik parameters to conduct the alignments: -hs 15 -mmp 0.05 -mhp 100 -act 26 -p 8 -bw 51. We discarded all reads that Mosaik mapped to the genome and kept only those that could not be mapped. We then used BLAT (version 3.4) [30] to map the latter reads. We ran BLAT using two sets of parameters: -fastMap and -oneOff==1. Finally, we detected CNV breakpoints with custom Perl scripts that parse the BLAT output and identify the split-read signature detailed in the Results section.
As discussed in the Results section, we applied a series of filters in an attempt to minimize the number of false-positive calls. One of the filters applied was the removal of all CNVs supported exclusively by reads that could be PCR duplicates; these were defined as reads with the same exact start position but that could vary in their end position. Because of this filter all seven putative tandem duplications identified by our pipeline were excluded from the final CNV dataset. Another filter was the exclusion of all CNVs where at least 80% of the mutated sequence mapped to a known TE (TE annotation downloaded from FlyBase [47] release 5.29). After CNVs were identified, we re-aligned all supporting reads once again to the reference D. melanogaster genome using Clustal [31, 32] and those were the alignments used to classify the CNVs into the four classes of breakpoints. The CNV calls were polarized using the syntenic alignments between D. melanogaster and D. simulans [47]. If a deletion is called in one of the nine sequenced genomes but is also present (with similar breakpoints) in the D. simulans genome, then the most parsimonious explanation is that the variant is actually an insertion in the reference D. melanogaster genome (Additional file 3, Figure S3). Similarly, if an insertion is called in one of the nine genomes but a similar insertion is found in D. simulans, then the most parsimonious explanation is that we are detecting a deletion in the reference D. melanogaster genome (Additional file 3, Figure S3). CNVs were annotated (as exonic, intronic, and intergenic) using release 5.33 (retrieved from FlyBase [47]).
Evaluating the contribution of nearby sequences to the formation of de novo insertions
We used standalone Blast [48] (ncbi-blast-2.2.25) to identify stretches of high sequence identity between de-novo insertions and its neighboring sequences (for the window sizes defined in Results). We generated the random control sequences using BEDTools (shuffleBed; version 2.13.3) [49].
Comparison of CNV breakpoints identified in Drosophila and human genomes
Table 1 compares the types of CNV breakpoints identified in Drosophila and in two independent human datasets: one generated by Conrad and colleagues [19] and the other by Kidd and colleagues [20]. Both surveys of human CNVs included the identification of small tandem repeats (variable number tandem repeat (VNTR)) and of variants associated with the movement of transposable elements. Because studying these variants was not an aim of this work, Table 1 only refers to breakpoints of deletions and insertions. We also excluded from the dataset generated by Conrad and colleagues the 13 deletions (out of 315) that were also associated with inversions. Finally, as discussed in the Results section we only consider microhomology when there are at least 2 bp of identical sequence present at the breakpoint. That meant re-classifying the breakpoints identified by Conrad and colleagues because they only required 1 bp to classify a CNV breakpoint as being associated with microhomology (detailed breakpoint information was made available by the authors as Supplementary Material) [19].
Evaluating the roles of NAHR and SSA
To test for sequence identity shared between regions within CNV coordinates and flanking DNA, three sequence databases were generated for both the 454 and microarray data, using the reference Drosophila genome (version 5.27): (1) CNV sequence; (2) 5' flanking sequence; (3) 3' flanking sequence. The 454 data provide precise CNV breakpoints and, based on these coordinates, we extracted 200 bp 5' and 3' of the CNV. The microarray data do not provide exact breakpoints, and for these data we defined the distal ends of the flanking sequences to be 500 bp 5' or 3' of the CNV coordinates. The proximal coordinates of the flanking sequences were set to extend 25% the length of the CNV 3' of the start of the CNV, or 25% the length of the CNV 5' of the end of the CNV. BLAT [30] (blatSuite.34) was used to search for sequence identity between: (1) 5' flanking sequence and the DNA within the CNV coordinates; (2) 3' flanking sequence and the DNA within the CNV coordinates; and (3) 5' flanking sequence and 3' flanking sequence. The data were filtered to return two datasets for each of these searches. The first filter was set to accept stretches of ≥30 bp that possessed ≥98% sequence identity; the second was set to accept stretches of ≥200 bp that possessed ≥95% sequence identity. The microarray results were further filtered to remove all 'self-hits' that resulted from the flanking sequences overlapping the CNV coordinates. Fasta files were generated for all sequences meeting the above criteria and were screened for repetitive sequences using RepeatMasker [50] (settings: abblast search engine, default speed/sensitivity, D. melanogaster annotations).
Identification of recurrent and complex CNVs in the tiling array data
We randomly selected 26 regions of the D. melanogaster genome that were identified by Emerson and colleagues as having deletions and that had been confirmed by PCR [5]. We screened these 26 regions in the same 15 natural populations analyzed by Emerson and colleagues. We identified recurrent CNVs by the presence of bands of different size (generated using the same pairs of primers) in different populations. We then sequenced these different bands by Sanger sequencing.
All statistical analyses were done using the statistical package R [51] and the application Rstudio.
Data availability
The Sanger sequences of the breakpoints of simple and complex CNVs initially identified using the tiling array data have been deposited in GenBank (KC138560-KC138678).
List of abbreviations
- CNVs:
Copy number variants
- FoSTeS:
Fork stalling and template switching
Homologous recombination
- LCRs:
Low-copy repeats
Microhomology-mediated break-induced replication
Microhomology-mediated end-joining
Non-allelic homologous-recombination
Non-homologous end-joining
Serial replication slippage
Single-strand annealing
- VNTRs:
Variable number of tandem repeats.
MC-M was supported by a postdoctoral fellowship from the Portuguese Foundation for Science and Technology (co-financed by POPH/FSE) and this work was supported in part by NIH grants R01 HG 003229 and R01 AI 064950 to AGC.
Authors' Affiliations
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Wanting to know what those who feed or have fed Taste of The Wild dog food think of it. I have a four month old ylm and he is pushing 50 pounds on Iams large breed puppy food. I have been told by a few people that the TOTW food is great, but would like to hear more of the experiences fro lab people.
Tried a search, but have to go through a lot of posts to find anything about it and then it does not say a lot about it. AT TSC it was very close in price with the Iams large breed food, but i think the bag held a little more.

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Spiral II Area Rug
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If you are close to Madrid, the kennel I posted does a good job of giving them exercise. Very clean and nice kennel
My approach is this: I use a trustworthy boarding kennel, and they feed, air, and clean up after my dogs. My dogs get ample time to air and move around - but they are not being handled, they are not retrieving, they are not playing with others' dogs at all.
I find when I return that my dogs are fine, may smell a bit bad, probably need a bath, and a good run. Within a day or two of my return, we are right back on a reset exactly where we were prior to my vacation. No bad habits, no injuries from a dog play gone wrong, No quirky stuff because someone "taught" them a new standard for "sit", "heel", etc.
The only way I'd consider allowing someone else to handle or train my dog is if that is specifically why they are getting my dog. An entirely different set of selection criiteria and qualification will be used. For vacation, I just want my dogs boarded safely, with no commands and NO TRAINING done while I'm away.
[QUOTE]At my vets office he is indoors in the airconditioned building. The vet has kids who work for him who take my dog out and play with him every day inside a fenced area. They love Scooter because he sits and plays fetch so he gets extra attention. He gets a free bath if he stays more than 3 days and while they have him they go ahead and give him his shots. This saves me a trip. He comes home happier and smelling better. All for $10.00 a day $140.00 for a 2 week vacation. Worth every penny.
I found the vets to be tons better than the trainers. I am not knocking trainers at all. They are busy and don't have a lot of time for a dog that isn't staying is all I'm saying.[QUOTE]
As with everything else in life, it depends. I used to work as a vet tech. One hospital I worked for had what I considered to be a very poor set up for boarding dogs. First, only dogs under 50lbs could be boarded since they were kept in cages, not kennels. For most dogs, this was only enough room to stand up and turn around in the cage, which they spent > 23hours in each day. Second, even though we were instructed to tell clients that we took each dog on 3 ten minute walks a day, truthfully it was only two walks a day and for only as long as it took the dog to pee and poop (which could literally be as short as 1 minute, never more than 5). Very few dogs got individual attention from any of the staff. Only "favorites" really got any special treatment and it was very little at that. There simply was not time to play with the boarding dogs and fulfill tech/secretary duties. Kenneling fees were no cheaper than local boarding facilities. The dogs were in a climate controlled setting, which is important during extreme temps, not so much in moderate temps. I would guess that a dog would be much more stressed in a tiny cage with air conditioning then a full kennel run in 40-80 degree weather.
So obviously, one must do a little research before using any arrangement. Based on my experience working for this particular vet, I would never take what is said as fact. Ask to see the boarding set up and if seperate staff is specifically hired just to take care of the boarding animals. If you can, ask to observe staff while they are walking the dogs to see how long each dog really gets outside. I'm sure many vets have adequate facilities to watch your dog, but not all and not everyone is upfront with their clients.
Agree w/boarding at a vet's or good boarding kennel. Unless of course you're already a client of a trainer that's willing to keep your dog short term. I had to board my big male Chesapeake when all my females including his mother and daughter came in season at the same time. A pro that had already trained a dog for me agreed to board him for a month. I asked if he could maybe get some retrieves while he was there; as the trainer had previously not been much of a fan of the CBR breed. He ended up staying there and getting his SH and MH.
Ask for references and tell people what you want. I found veterinary clinics mostly had better set ups for what I wanted than boarding kennels when researching 2 yrs. ago. I inherited a condo that has a no pets policy and is on a no dog beach in Naples, FL. I like to bring a Chesapeake for the 2-day drive, because I feel safer in the budget motels What I found in the boarding-only places is they seem to cater to foolish rich people with spoiled little drop-kick dogs: "cage free" boarding, group play time (((((shudder))))), fancy dog bed "suites" etc., anything to get doting doggie owners to part with major sums of money, starting at about $30-40 a day. The vet I found had knowledgeable staff, very nice a/c runs separate from where the sick animals are kept, and a big fenced airing yard that was spotless. My dog was well cared for, aired multiple times daily and even though this vet was located in a high rent district, it only cost $10/day. I love my dogs, but they are not children who need to play with others nor do they need fru fru fancy beds and piped in music!
Julie R., Hope Springs Farm
Chesapeake Bay Retrievers since 1981


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Web Stories
Martin Amis to read from work, Nov. 28
Posted November 16, 2001; 09:49 a.m.
novelist Martin Amis will read from his work at 4:30 p.m. Wednesday, Nov. 28, in the Stewart Film Theater, 185 Nassau St.
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His reading is sponsored by the Althea Ward Clark Reading Series in the Creative Writing Program .
Contact: Marilyn Marks (609) 258-3601


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Aid Worker Diaries: "I was struck by the strength and resilience of the people in Ishinomaki."
9 March, 2012
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On 11 March 2011, Japan experienced one of the worst natural disasters in its history. The Great East Japan Earthquake claimed nearly 16,000 lives and destroyed some 128,000 homes and buildings. More than 3,200 people are still missing.
In the hours following the disaster, a joint OCHA/UN Disaster Assessment Coordination (UNDAC) team was dispatched to Japan. The team helped the Government manage offers of humanitarian assistance, assisted in search and rescue efforts, and reported on the humanitarian needs.
Masaki Watabe, who was recently appointed Head of the OCHA Kobe office, talks about his recent visit to the coastal town of Ishinomaki in Miyagi Prefecture – one of the worst affected areas.
Everyone has events in their lives that were so shocking that you always remember exactly what you were doing when you heard the news. For me, one of those events was the 9.0 magnitude earthquake and tsunami that struck Japan's northeast coast on 11 March 2011.
I was living in New York and woke up to see the terrible images on the television – this was the first mega disaster that the world watched unfolding before their eyes. My family and I watched in horror.
A year has passed and I am now living in Japan working for OCHA. Together with other partners we are mobilizing this country's tremendous resources - wisdom, experience, and institutions – to help other countries respond better to natural disasters and humanitarian crises.
A few days ago, I visited the fishing town of Ishinomaki in Miyagi Prefecture for the first time. In that town, 3,800 people lost their lives or are still missing. So much was lost all at once – family members, friends, houses, schools, jobs and workplaces. For most of us this is unimaginable.
I visited the areas of Ishinomaki close to the ocean - now they are just vast empty spaces. Many of the damaged houses have been torn down. I felt as if I was standing at 'ground zero.' Much of the debris has been cleared and is now piled up waiting to be processed. To date, only 5 per cent has been processed and it might take years to finish.
I went up to the top of Hiyoriyama hill, where many people fled to safety on that day, and quietly looked down at the empty land below. I was there with a man who had survived the disaster and he said to me, "I must live - because something let me live."
Today, more than 342,000 people remain displaced from their homes in Japan and are living in temporary housing, including more than 34,000 people in Ishinomaki alone. The temporary houses have fairly basic equipment – by Japanese standards – including a refrigerator, a rice cooker and an electric kettle.
Despite the chilly snowy weather, the people living in these houses gave me a warm welcome. I joined the 'tea salon' activities organized by volunteer organizations, where we built some flower boxes as Spring is around the corner, and decorated lanterns for the 3/11 anniversary. The men looked like they were enjoying the carpentry work, as it was meaningful and gave them something to focus on. The women chatted with each other about their grandchildren, hobbies, cooking, and their survival stories. The elderly ladies talked about their memories as well as their lives now - and the future. I did not ask many questions. Instead I listened.
I recognized the same sense of sorrow in people that I met after natural disasters or conflicts in other parts of the world. As well as dealing with anxiety and loss, the displaced families are also struggling to restore ties with others, and trying to rebuild their communities. Residents of the pre-fabricated houses were selected by lottery, and houses were allocated to households at random.
Although people address psycho-social needs in different ways across countries and cultures, there is no doubt that psychological health is an essential part of living a meaningful and dignified life. Therefore, meeting new neighbours through occasions like the 'tea salon' is important for them to feel connected.
I was struck by the strength and resilience of the people in the temporary housing, especially the elderly ladies who said they were grateful to have everything they needed. I heard how much they appreciated the generous support, but they also stressed that they do not want to become dependent on this aid. The people of Ishinomaki want to give back the support they have received – perhaps during future disasters in Japan or elsewhere.


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As a population, we are living longer because of healthier lifestyles, new medical technology, and drug treatments. These medical advances enable us to live with a mental or physical condition longer than ever before. A 65 year old individual today is expected to live well into his/her late 80's. Because we are aging and living longer, our ability to perform normal activities of daily living could be hindered due to a medical issue. Long term care services assist an individual with everyday activities such as eating, bathing, dressing, and mobility that are hindered because of a medical or mental condition.
The Indiana Long Term Care Insurance Program (ILTCIP) is an innovative working partnership between the State of Indiana and private long term care insurance companies. Indiana has taken the lead in helping its residents protect their hard-earned life savings from the high cost of long term care by promoting the awareness of long term care.
All long term care insurance policies available from insurance companies are not the same. Policies are approved by the Indiana Department of Insurance as meeting required state statutes. "Partnership policies" offer the consumer additional benefits for their long term care needs and protection for their savings. In addition, Partnership insurance policies qualify for a state tax deduction helping Hoosiers to protect even more of their savings.


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You've just finished a tennis match/drum circle/swim/ball hockey game/Frisbee session/baseball practice at Trinity Bellwoods Park and now you want a continuous street to take you North beyond Bloor Street.
Well, you could try Bathurst with its awkward intersections, streetcar tracks and speedy drivers. You could head over to Ossington, a street with more potholes than parking spaces. Or, you could simply head North on Crawford, a one-way street with a gentle uphill grade and even a touch of cycling infrastructure.
Although there are blue bicycle signs along Crawford, you won't find this residential street listed as part of Toronto's Bikeway Network.
While not an official signed route, there is signage alerting drivers to the presence of cyclists.
Just north of Dundas, Crawford is spacious and their are speed bumps and stop signs to help slow motorists who are also supposed to keep their speed at or below 30 km/h.
Although the extra space on this street often encourages illegal parking:
A slight uphill and a few minutes later and you reach a controlled intersection at College Street:
If you're turning left, stand on the dots:
There are no dots here, but if you're going straight this may set the traffic signals in motion. I've never waited here more than a few seconds:
Just north of College, Crawford takes a twisty turn lined with street parking. The right curb side has uneven manhole coverings, so take the lane:
Here's where it gets strange. Where do I go?
If you can spot the cyclist on the left of the above photo, that's where Crawford continues. Head right, and you'll end up on Montrose. There's a blue circle bicycle sign hidden, too:
A few short minutes later and you'll reach Harbord where you can connect to bicycle lanes. Should you want to go straight through this is where Montrose one street over to the East is a better option. At Harbord you have a stop sign, yet the east/west traffic does not. Depending on the time of day you may have to wait awhile to get across:
Watch out for right hooks while you wait:
And then carry on:
And once again, prepare to wait at the even busier Bloor Street:
As bike lanes continue to be a major issue in the upcoming Toronto mayoral elections, it is important to consider our entire network. Bike lanes are needed on major arterial roads and bicycle-friendly infrastructure is needed on our secondary street routes. That includes signaled crossings, something that would improve Crawford and earn it a permanent role in Toronto's Bikeway Network.


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THE Poet and the Thinker sought for truth.
God bent and held a hand to either.
To the poet he put out his hand from a cloudless vault of blue; the Poet saw it, and climbed.
To the Thinker God stretched his hand from the heart of a mighty cloud; the man looked up and saw it move: he mounted.
On far‐off mountain sides they laboured, looking upwards.
Then he who looked into the blue, cried: "Brother, you are wrong! What lies above you is but dark cloud; reach it—you will find it cold mist. In it you will wander for ever. Over me in the blue sky is that which calls me; I rise to it!"
The Thinker answered: "Fellow, you are dazed. The sun has shone too long upon your head. What lies above you is an empty vault of blue. Enter it, you will find it empty space; you will grasp—air! Over me in that dark storm cloud lives that which calls me: when the lightning flashes and the thunder rolls and the cloud is riven, I see illuminated that which beckons. I mount to it."page: 130
The Poet cried—"Fool!"
The Thinker—"Blind!"
They both mounted.
At last, when they were very tired, they reached their mountain summits.
God bent, and took his Poet in his left hand, and his Thinker in his right, and laid them in his breast. When they awoke, they were side by side upon the heart of God. One whispered, "By the left hand, I!"; the other, "By the right!" ... and they heard the truth beat.


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Yet another Snow! Once Upon a Time lacks the stakes of the other Snow White stories, and Ginnifer's wigs are pretty awful. But where we have hope is that Ginnifer plays both the traditional Snow and her modern-day counterpart who doesn't remember her past life. So maybe we'll get some real character development beyond what we saw in Disney.
Mia will always be the OG for us, since many of us grew up watching Disney's slightly whitewashed version of Meg Cabot's snarky Princess Diaries series. What's funny is that Love and Other Drugs saw Anne with much the same haircut, but it was so much more attractive the second time around. But really, playing the sneakers-clad, uncouth big-city princess was a great breakout role for her.
Another classic! Though she gets kidnapped early in the movie, Buttercup redeems herself by standing up against her captors -- and later the prince who wants to marry her -- and staying true to her love Westley (Cary Elwes).
This movie is an instant classic with my friends because it's a pretty solid romantic comedy, but still pretty cheesy. Julia Stiles followed up her evil man-hating Katarina in 10 Things I Hate About You with a character who's equally motivated but actually open to love--only, after she graduates from med school. (Sadly, Luke Mably does not hold a candle to Heath Ledger.)
This was one of my favorite of the modern Disney princesses; the animation was lovely, and Amy Adams mocking the naivete of fairytale princesses before her was hilarious. She's not a bad singer, either, especially when accompanied by New York City's rat and roach populations.
We have to guess that Fiona is based in part on Buttercup. While none of the princesses we know in pop culture are especially helpless, there's an extra depth to her character because Fiona is the recipient of an awful curse: Every night she turns into an ugly ogre.
Hallmark's Prince William/Kate Middleton movie was basically a rip-off of The Prince & Me, so we're going to skip on ahead to the much more interesting Lifetime version...
Catherine: A modern princess who won her prince by prancing around onstage in a see-through swimsuit/lingerie piece.
I thought to myself, "Audrey Hepburn must have played a princess at one point in her career." And voila! Here she is as the sheltered Princess Ann, who escapes her country's embassy to explore Rome with an expat reporter (Gregory Peck).
The original hubbub about Disney's first black princess was that the company had considered making her a slave girl, only to have people of color fight back against what they thought was racist storytelling. But now that I've read through the Wikipedia article, it sounds like Tiana only becomes a princess once she marries the frog prince? Then again, that would put her on equal footing with many of the white princesses in this gallery. Equality!
Here's another radical reimagining of a well-known fairytale: Bella Swan actually grows a pair and picks up a sword to fight the evil queen for the right to her throne!
Lily Collins' version of Snow White may be less adept with a sword--anyone would be, if they didn't have Chris Hemsworth teaching them--but she's still got fire in her.
Yet another Snow! Once Upon a Time lacks the stakes of the other Snow White stories, and Ginnifer's wigs are pretty awful. But where we have hope is that Ginnifer plays both the traditional Snow and her modern-day counterpart who doesn't remember her past life. So maybe we'll get some real character development beyond what we saw in Disney.
The natural redhead will either go the drastic route of chopping off her hair and dyeing it blond, or wearing a wig.
Much like My Week with Marilyn, the Diana profile in Caught in Flight won't be all rosy and nostalgic: indieWIRE says that she will be depicted "as a damaged person who stalks the doctor after he ends the affair."


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Everything you need to know about today's media coverage of Israel and the Mideast.
Will Big Media take any interest in legal rulings chipping away at the myth of Mohammed al-Dura? Is the "Start Up Nation" phenomenon rubbing off on Palestinians? And who is really responsible for Gaza's continuing fuel crisis?
Join the Media Cheat Sheet Page on Facebook.
Israel and the Palestinians
• Following a French court's acquittal of Dr. Yehuda David, YNet looks at some of the other legal cases stemming from the al-Dura video.
This is not the only slander trial surrounding the al-Dura case. A series of trials revealed an abundance of contradicting testimonies, and evidence trying to ascertain whether al-Dura was killed by IDF fire or Hamas cross fire. However, rulings are always limited to the question of slander, leaving the mystery of al-Dura open for debate.
See also Lilac Sigan's must-read reaction asking, Where's the media?
I don't blame the boy's father for lying, by the way. If it were me that had to choose between lying, or telling the truth and being attacked with axes and knives by terrorists again, I'm pretty sure I'd do the same. But while the poor father's motive for lying is clear — what's the motive of the western world to keep silent about the truth, and prefer to keep believing the myth?
Sadly, these are the rules of our allegedly truthful world. The myth was aired all over the world and burned into the collective memory. It will live forever, and who knows how many others will be "inspired" with hatred as a result. And what about the real story? For some crazy reason — no one is interested in reporting it. None of the major news channels that reported the incident 12 years ago felt necessary to report the ruling of the French supreme court. I guess it will remain our little secret, then. It kind of makes you wonder — maybe all we really want is a good story with a hero? And once we get it — please, don't confuse us with the facts.
• Dan Margalit comments on Khader Adnan's hunger strike. The Islamic Jihad member's stunt is now at day 65.
Ashton and others like her, here and around the world, have demanded that Israel minimize its use of administrative detention. One can understand where she is coming from, and this should be Israel's objective too. But we must beware of the Islamic Jihad prisoner's objective: He is trying to force Israel to refrain from one of the integral and essential aspects of the war against terror. If he wins, there will be no more administrative arrests . . .
Israel should make it very clear to the world that three principles will guide its approach toward Adnan and others like him: Israel will use administrative detention as infrequently as it possibly can; it will fight attempts to force it to abandon a key aspect of the war on terror, despite efforts by Adnan, his father and a handful of journalists to undermine the country's security; and at the same time, Israel will forcibly prevent Adnan from dying.
• Gaza's fuel crisis ain't over yet. Maan News says Egyptian fuel deliveries hit a snag you could've seen coming:
Egypt wants to stop the passage of fuel through tunnels under the border between the countries, but the official Rafah terminal is not equipped for goods transfers and its development is restricted by an agreement between Egypt, Israel and the West Bank-based Palestinian Authority.
The only alternative route while this agreement is being renegotiated is for the fuel to cross into Israel, and then back into the Gaza Strip . . .
Unfortunately for the people of Gaza, this Haaretz update says Hamas and the PA are balking at the latter option.
• Newsweek calls Beitar Jerusalem onto the carpet over the soccer team's lack of Arab players:
"It's hard to explain the policy as anything but racism," says Yoav Borowitz, an Israeli journalist who writes regularly about football.
Supporters of the team have a more nuanced explanation. They say the ban is bound up with Beitar's history and with tensions in Jerusalem, a city where Arabs and Jews live mostly in their own segregated neighborhoods (Israeli Arabs make up 20 percent of Israel's population) . . .
But people who know Beitar from the inside say the managers fear that any change would prompt a backlash among fans. "Even if the racists are a small minority, they would cause a lot of trouble in the stands if the team suddenly put an Arab on the field," said one person affiliated with the team who did not want to be identified.
• Is the "Start Up Nation" phenomenon rubbing off on the Palestinians? According to the Christian Science Monitor, West Bank entrepreneurial geeks are hip:
Until now, the primary Palestinian contribution to technology has been outsourcing programmers and engineers to firms in the United States and Israel, including Google and Cisco Systems.
But these new entrepreneurs want to do more. They want to create companies based on their own ideas and hire people to implement them. Already their ventures range from smart phone apps to Web design.
Crucially, the community is now beginning to attract investors.
• Haaretz: Bibi's peace proposals at January's Amman talks are remarkably similar to Kadima's plan.
• Canadian Foreign Minister John Baird is tougher on the Palestinians than his speechwriters. Now you know:
Mr. Baird ended up delivering a much tougher address than envisioned by his speech writers, one that unequivocally emphasized Canada's support for Israel – a position for which he makes no apologies and which has generated much criticism of the Harper Conservatives.
Copies of the draft texts of the speech, obtained under the Access to Information Act, show Mr. Baird used a radically reworked text when he represented Canada for the first time at the General Assembly on Sept. 26, 2011.
Iranian Atomic Urgency
• The NY Times assesses what an Israeli strike on Iran would entail. We're talking about four primary targets, three possible flight routes, and at least 100 planes making a round trip of some 2,000 miles.
• Staff-eds in The Guardian and Irish Times urge conflict avoidance with Iran.
Spring Winter
• A Syrian rebel tells CNN their uprising is an an "orphaned revolution." With no international support, people are turning to Allah.
• A correction's certainly warranted. But who in Australia wants to speak up for Bashar Assad and protest this Sydney Morning Herald headline?
For more, see yesterday's Media Cheat Sheet.


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To perform a search, enter a keyword, phrase or item number in the search field and click "search."
We offer two ways to locate information:
The white varietals of Parellada, Chardonnay, Macabeo and Xarel-lo are grown in Jaume Serra's vineyards situated in the Alt Penedés, a sub region of the Penedés, and are used to produce the area's famous sparkling wines, called "Cava". By law, Cava must be made in the Methode Champenois or Champagne Method. Due to European regulations prohibiting use of that term, Cava is now labeled as Methode Tradicional, which has the same requirements with an added stipulation that cava must age for a minimum of nine months on the lees before release. After researching local yeasts, the Jaume Serra winery has taken the extraordinary step of importing their yeast from Champagne, France in order to impart the finest quality to their Cavas.


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I've never seen so many new posters on Potus Place, the best kept secret this side of the White House. If you want a good debate, you're in the right place.
The retriever world has many bleeding -heart socialist and though we know we can't reform them, we can certainly minimize thier messages here at Potus Place.
Step on up, say your piece and get ready to defend it!
P S We almost had a thread here turn to dog training. This is not permitted! We had to remind that person that dog training belongs on the other page.
Thanks Chris, I couldn't help myself!


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Pillsbury® Grands!® Jr. Golden Layers® biscuits provide a simple addition to this spicy pork and vegetable casserole that's baked with Progresso® chicken broth - perfect for dinner.
One pound of boneless pork shoulder can be substituted for the pork tenderloin.
Chimichurri originated in Argentina as a sauce or marinade for grilled meats. It is typically served as an accompaniment, but we've used the ingredients to flavor this casserole.
© 2017 ®/TM General Mills All Rights Reserved


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What is a complement?
|Definition 1
Traditionally, a complement is a constituent of a clause, such as a noun phrase or adjective phrase, that is used to predicate a description of the subject or object of the clause.
|Here are some kindsof complements under this definition:
|Definition 2
In generative syntax, a complement is a phrasal or clausal category which is selected (subcategorized) by the head of a phrase.
A selected, or subcategorized, phrase is obligatory, as contrasted with adjuncts, which are optional. For instance, the direct object of a transitive verb is obligatory and therefore a complement, whereas adverbial modifiers are generally optional, and therefore non-complements. However, the distinction is not always clear, particularly for oblique arguments. Neither is the distinction clear in languages in which complements can be freely omitted if they are understood from the context. Omission must be distinguished from pronominalization; pronouns may generally be considered to be complements. However, in some languages pronouns have been grammaticalized as verbal affixes, in which case the question of whether they are complements or not becomes a theory-internal question. (See Baker 1996, chapter one for discussion of this issue.)
The complement/ adjunct distinction cross-cuts the core/ oblique distinction, since there are obliques which are complements, and other obliques which are adjuncts. Also, while the subject of a clause is often considered a core argument of the verb, it is not normally considered to be a complement. This is because in most (perhaps all) languages, the subject appears to be a clause-level constituent, rather than a constituent of the verb phrase. However, it should be noted that this argument presupposes that the verb and its object belong to the same phrase-level constituent, while the subject is outside that constituent, an analysis which leaves the status of subject and object in VSO languages unclear.
|Examples (English)
|Nonexamples (English)
|A complement is a kind of
Mish 1990 269
Crystal 1985 60
Quirk, Greenbaum, Leech, and Svartvik 1985 55, 741–742
Andrews 1985: 89–92
Context for this page:
This page is an extract from the LinguaLinks Library, Version 5.0 published on CD-ROM by SIL International, 2003. [Ordering information.]
Page content last modified: 5 January 2004
© 2004 SIL International


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"[I] am a very happy man doing what is in effect my hobby for a living, i.e. writing stories for children."—Dick King-Smith
Dick King-Smith grew up in Gloucestershire, England. He lives with his wife in a small 17th-century cottage, three and a quarter miles from the house in which he was born.
I was born in 1922 and was brought up in the countryside, surrounded by pet animals of one sort or another. I was first attracted to the girl I was later to marry through my admiration of her skill (in contrast to my own haphazard husbandry) as a breeder of parakeets. I was thirteen at the time, and she was twelve. It hasn't worked out too badly, as we have been married for nearly sixty years, three children and ten grandchildren to the good.
I was educated at a preparatory school and then at Marlborough College, a boarding school for boys, where I was good at sports (but not very good) and showed reasonable intelligence (though not very great).
If I had an ambition, it was to be a farmer. But then along came the Second World War, and so I took the king's shilling—that is to say, I enlisted as a soldier. I served as a platoon commander in the Grenadier Guards, beginning my active service at Salerno with the Allied invasion of Italy in September 1943 and ending it eleven months later when a German paratrooper threw a British hand grenade at me in the middle of an Italian forest and filled me full of holes.
My career in farming began in 1947, and though I think I was a decent stockman, my business acumen was extremely low. It was difficult to farm at a loss in those days, but I managed to lose money without fall every year from 1947 to 1967, when the bank manager called a halt.
Six months of selling aluminum-asbestos firefighting suits, boots, and helmets (I didn't sell many) were followed by three and a half years as a time-and-motion man in a shoe factory (I never really understood the mathematics). Then I trained as a teacher and took a degree from Bristol University at the age of fifty-three, reading English and philosophy. I did well in philosophy even though I hadn't the faintest idea what it was all about, and still haven't.
Next came seven years as a teacher in a small village elementary school. I began with eight-year-olds, but it soon became plain that my always shaky grasp of the principles of arithmetic was not adequate for people of that advanced age, so I ended my teaching career with children of five and six.
By this time I had written my first four stories, and now—with time of my own—books began to spring up like mushrooms. In addition, I landed several little jobs as a presenter on children's television, where I met a whole lot of delightful people and animals and much enjoyed performing in front of the camera.
I'm still turning out books hand over fist—now nearing a hundred in print in the United States, England, and Europe—mostly about animals: farmyard fantasy, I suppose you could call it, often about pigs, my special favorites. I enjoy writing for children so much, and meeting them, and knowing (because they tell me) that they get enjoyment from what I do.
I'm happy, and so are my wife and my children and my grandchildren and the bank manager. I'm a lucky man.
The Gallant Pig
—An ALA Notable Book
—A Boston Globe–Horn Book Honor Book
—A Horn Book Fanfare Honor Book
—An IRA/CBC Children's Choice
—An NCTE Teachers' Choice
"An animal fantasy which will inevitably be compared to Charlotte's Web. . . . Combines a robust pleasure in the smell and feel of rural surroundings with a humorous affection for all living creatures . . . a splendid book."—Starred, The Horn Book Magazine
"Thoroughly engaging . . . chipper dialogue, generous helpings of humor and a lickety-split plot add up to an amusing chapter book."—Publishers Weekly
"Amusing . . . a fine choice for early chapter-book readers."—School Library Journal


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