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High levels of RNA-editing site conservation amongst 15 laboratory mouse strains
Genome Biology volume 13, Article number: r26 (2012)
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Adenosine-to-inosine (A-to-I) editing is a site-selective post-transcriptional alteration of double-stranded RNA by ADAR deaminases that is crucial for homeostasis and development. Recently the Mouse Genomes Project generated genome sequences for 17 laboratory mouse strains and rich catalogues of variants. We also generated RNA-seq data from whole brain RNA from 15 of the sequenced strains.
Here we present a computational approach that takes an initial set of transcriptome/genome mismatch sites and filters these calls taking into account systematic biases in alignment, single nucleotide variant calling, and sequencing depth to identify RNA editing sites with high accuracy. We applied this approach to our panel of mouse strain transcriptomes identifying 7,389 editing sites with an estimated false-discovery rate of between 2.9 and 10.5%. The overwhelming majority of these edits were of the A-to-I type, with less than 2.4% not of this class, and only three of these edits could not be explained as alignment artifacts. We validated 24 novel RNA editing sites in coding sequence, including two non-synonymous edits in the Cacna1d gene that fell into the IQ domain portion of the Cav1.2 voltage-gated calcium channel, indicating a potential role for editing in the generation of transcript diversity.
We show that despite over two million years of evolutionary divergence, the sites edited and the level of editing at each site is remarkably consistent across the 15 strains. In the Cds2 gene we find evidence for RNA editing acting to preserve the ancestral transcript sequence despite genomic sequence divergence.
The adenosine deaminase acting on RNA (ADAR) family of enzymes is capable of modifying adenosine residues to inosines [1]. Inosine is interpreted by the transcriptional machinery as guanosine, and thus appear as A-to-G mismatches in cDNA sequences. The ADARs bind to double-stranded regions of RNA and can modify multiple neighboring adenosines until a tolerance level is reached and the structure of the RNA becomes destabilized [2]. RNA editing in coding regions can lead to alteration in protein function and increased transcript diversity. The best known example of this is editing of neural serotonin receptor HTR2C gene transcripts, which are edited at five sites in close proximity to each other [3], thereby producing a diverse repertoire of 28 mRNAs and 20 protein isoforms [4]. Isoform composition and editing frequency are lower during early developmental stages and gradually increase with age until adulthood [3]. RNA editing can also induce alternative splicing; for example, the enzyme ADARB1 edits its own pre-mRNA, which leads to expression of a protein with diminished catalytic activity [5]. The functional consequence of RNA editing of non-coding sequence is not well understood, although it has been suggested that editing of UTR sequences may be associated with their nuclear retention [6]. RNA editing occurs in all metazoans, but the level of editing differs substantially across species. It has been estimated that one nucleotide in every 17,000 is edited in the rat brain transcriptome [7], while in the human brain transcriptome the number of RNA editing sites is thought to be an order of magnitude higher [8, 9].
Large-scale efforts to generate cDNA sequences, and to annotate reference genomes, have greatly facilitated the discovery of RNA editing sites in the transcriptome of several species. Zaranek et al. [9] curated several tens of gigabases of human and mouse cDNA sequence to derive one of the most comprehensive surveys of RNA editing to date. More recently, the advent of second-generation sequencing technologies, and the development of the RNA-seq method, has made it possible to sequence the entire transcriptome [10]. Recently, several authors have attempted to use RNA-seq data to discover RNA editing sites. Li et al. [11] predicted approximately 10,000 RNA editing sites from RNA-seq data from human B-cells and reported all 12 possible base changes. Bahn et al. [12] used a different computational methodology to report a similar number of RNA editing sites in the transcriptome of a human glioblastoma cell line but found that the vast majority of edits (approximately 70%) were of the A-to-I type. It is clear from these discordant results that the accurate identification of true RNA editing sites is computationally challenging and great care must be taken to identify and remove artifacts such as alignment errors.
In this paper, we present the first comprehensive survey of RNA editing in the mouse genome across a diverse set of 15 mouse strains that together represent all of the major laboratory mouse lineages. To achieve this, we start with detailed knowledge of the genomic variation across the strains [13] and combined this information with single nucleotide variant (SNVs) called from RNA-seq data to generate an initial set of candidate edit sites, which were then systematically filtered to produce a highly accurate set of RNA editing sites that was dominated by A-to-I edits (Additional file 1). Using this filtered set of RNA editing sites, we investigate the pattern of RNA editing and used a knowledge-driven approach to significantly improve the sensitivity of the catalogue, especially in regions where there are clusters of RNA edits within the transcriptome. Our analysis produces one of the most comprehensive pictures of the extent of RNA editing in a vertebrate genome with an estimated false discovery rate (FDR) of between 2.9 and 10.5%. While most RNA editing sites lie outside of protein coding regions, we identified non-synonymous edits that contribute to transcript diversity and possibly protein diversity. Our analysis suggests that RNA editing in protein coding sequence is more commonly found in receptor and ion transport functional classes.
Identification of cDNA/genome mis-match sites
RNA was extracted from the brains of mice from 15 strains (129S1/SvImJ, C57BL/6NJ, C3H/HeJ, A/J, AKR/J, DBA/2J, LP/J, CBA/J, BALB/cJ, NZO/HlLtJ, NOD/ShiLtJ, CAST/EiJ, PWK/PhJ, WSB/EiJ and SPRET/EiJ). cDNA was then generated and sequenced [ENA:ERP000614], and the resulting reads (paired end 76 bp reads) were aligned to the mouse reference sequence (mm9) with BWA [14]. At least two biological replicates were generated for each strain. SNVs were called using samtools/bcftools [15] as described in the Materials and methods. The initial set of variant calls consisted of 304,817 candidate RNA editing sites and contained a large proportion of non A-to-G mismatches (Figure 1a). Manual inspection of these calls revealed that the vast majority of these sites could be attributed to the misalignment of short read sequences (Figures s1 to s3 in Additional file 2) primarily due to the placement of reads to multiple sites and mis-alignments around splice site junctions.
The reads localized to candidate RNA editing sites were then realigned in a splice-aware manner using exonerate [16] and all sites with reads that were not uniquely mapped were removed, resulting in a smaller set of candidate editing sites totaling 98,061 positions. Additional filters were then used that required a minimum cumulative depth of 10 reads per strain, and sites where we observed any mismatch in the genomic reads at a position were removed. To avoid artifacts from sequencing errors or possible low-level contamination, we also required an RNA editing site to be observed in at least two biological replicates of a strain. In addition, we discarded sites where reads with mismatches were biased in orientation (Figure s2 in Additional file 2). As indicated above, the major source of systematic error stemmed from the mis-alignment of reads spanning splice sites. Two filters, End Distance Bias and, to a greater extent, Variant Distance Bias, which evaluate biases in the position of mismatches within reads, proved extremely effective at removing these artifacts (Materials and methods; Figures s3 and s4 in Additional file 2). Following the application of these filters the set of RNA editing sites was composed of 5,579 positions, of which only 2.4% were non A-to-G or T-to-C mismatches (Figure 1b). Figure s5 in Additional file 2 shows the filtering steps that we applied and the effect of each filter on the number and type of RNA editing sites in the dataset.
Call set validation
We selected a random set of 611 calls from both the filtered set of 5,579 RNA editing sites and the much larger set of unfiltered candidates and validated these on the Sequenom platform [17] by genotyping genomic DNA from C57BL/6NJ and cDNA from each of the sequenced strains (Table s1 in Additional file 3). Approximately 30% of the assays yielded no signal or a signal that was inconsistent with the genomic DNA control and were excluded from further evaluation. Most of the failed assays appeared to be the result of Sequenom signals derived from multiple loci or a mixed PCR product. Comparison of the results from Sequenom assays to the calls made from the RNA-seq data revealed a discrepancy of 10.5%. Since this value was calculated as the number of sites that were not confirmed by Sequenom genotyping divided by the sum of the unconfirmed and confirmed sites, the figure of 10.5% is likely to reflect the upper boundary of the true FDR. Furthermore, a significantly higher proportion of sites where the editing level was less than 20% of transcripts did not validate by Sequenom genotyping. After manual inspection of these sites in the RNA-seq alignments we find no obvious systematic calling bias, suggesting that the sensitivity and specificity of Sequenom genotyping is, in these cases, lower than that of RNA-seq.
Further manual inspection of the non A-to-G edits in the refined call set revealed that the T-to-C mismatches were largely the result of uncertainties in the assignment of transcripts to a strand on the mouse assembly, or were the result of calls made in antisense transcripts. For example, the cluster of A-to-G mismatches (forward strand) that overlap the Zscan30 gene (reverse strand) appear as a novel form of T-to-C editing. However, the presence of another gene, Zfp397, and the Rpl19-ps7 pseudogene on the forward strand just upstream of Zscan30 suggests that antisense transcription or missing annotation are more likely explanations for the editing pattern at this site (Figure s6 in Additional file 2). The vast majority of the other non A-to-G or T-to-C mismatches could be explained as obvious alignment artifacts or low-quality mapping regions. Nineteen of the non A-to-G editing sites were amplified from cDNA and subjected to Sequenom genotyping. All were confirmed as false positives. Thus, based on this result we find that virtually all non A-to-G or non T-to-C mismatches are false positives. Assuming that the error rate for all types of mismatches are comparable, we estimated that the FDR of our call set is 2.9% (calculated using the formula k × s/N, where k is number of non-A-to-G and non-T-to-C calls and N is the number of all calls. The factor s = 12/10 increases the FDR estimate in order to account for errors in A-to-G and T-to-C bins). Interestingly, computational analysis followed by manual inspection of our final call set revealed only three non A-to-G edits that could not be explained as alignment artifacts, including a non-synonymous coding C-to-T mismatch in the Mfn1 gene at chr3:32,460,397 (Figure s7 in Additional file 2), which was validated by Sanger sequencing (Figure s8 in Additional file 2) and is compatible with the known C-to-U form of editing.
Knowledge-driven extension of the filtered set
As the calling procedure made no assumptions about the nature of editing, we were able to show that adenosine deamination is the primary form of RNA editing in mouse. In order to obtain a high-confidence set of calls, our initial calling filters were quite strict and possibly removed many true edits. Having established that most of the single-nucleotide changes in the transcriptome are driven by ADARs, we extended the set by taking advantage of the known property of ADAR editing that often modifies multiple bases in close proximity to each other. Any mismatches within 10 bp from an RNA editing site were iteratively added to the pool of calls along with clusters of eight or more mismatches of the same type within a window of 20 bp. All of the variants identified using this approach were A-to-G substitutions, and the number of RNA editing sites was increased to 7,389 (Figure s5 in Additional file 2). We tested 23 of these clustered edits and all were confirmed by Sanger sequencing (Materials and methods; Figure s9 in Additional file 2).
We next compared our extended set of 7,389 RNA editing sites and our initial candidate set of calls (304,817 sites) to RNA editing sites predicted in previous studies [9, 18, 19]. We found sequence coverage for 38% (313) of the sites predicted by Neeman et al. [18] and called 23% of these sites in our extended call set. When we compared our calls to the set described by Rosenberg et al. [19], 28 sites were covered by sequence reads in our study but none of these sites were present in the initial set of candidate RNA editing calls we produced. Manual inspection of all 28 positions in our mouse transcriptome data failed to provide support for 24 positions as being subject to RNA editing. Of the four remaining, one position at chr15:99,239,051 shows clear evidence of RNA editing but was inadvertently filtered out of the extended call set because of a random base mismatch in genomic sequence at this position. There were also three other sites (chr2:121,978,638, chr3:119,135,669 and chr3:144,259,976) where occasional C-to-T mismatches were visible, but these occurred with a frequency that was too low to make a confident call that RNA editing was operative at these positions. Another set we examined was from the study of Zaranek et al. [9]. We found an extremely low overlap of our calls with this set: only 5 out of 1,528 sites (0.3%) were confirmed by our study. Interestingly, 62% (944) of the sites in the Zaranek et al. study were found as SNPs in one of the 17 strains we sequenced, suggesting that these calls are heavily contaminated with genomic variants.
Editing rate and the sensitivity of variant calling
We observed substantial variation in the number of predicted editing sites across the 15 strains, with an average of 4,800 edits per strain (standard deviation 700) being called (Figure 2a). We reasoned that a certain proportion of this variability could be attributed to differences in the sequence depth of each transcriptome rather than biological differences between strains. Indeed, we found that for only 21% of RNA editing sites can we be certain that an edit was not missed due to low depth in one of the strains at a 5% significance level of a one-sided binomial test (Materials and methods). Using a 95% level of confidence that RNA editing was not missed due to low sequence depth, the average number of RNA editing sites per strain increased to 6,383 and the standard deviation decreased to 153.
In our extended set, we observed six to ten RNA editing sites per 100 kb of accessible reference sequence, or less than one inosine per 100 kb of transcribed sequence (Figure 2b). However, we suspected that the true editing rate may be somewhat higher as manual inspection of 28 editing clusters revealed that due to very low levels of editing, we may have missed around half of edited positions from our call set, particularly when these sites were tightly clustered. Nonetheless, based on this evaluation, missing sites account for just 4% of the total inosines owing to the fact that the editing levels at these positions is very low. These sites are extremely challenging to call computationally as they are difficult to distinguish from random sequencing errors. Higher sequencing depth would help provide support for these positions as sites of RNA editing.
Sequence context
Most RNA editing occurred in clusters of two or more sites (5,233 sites in 1,582 clusters) but 30% (2,156) of edits occurred at single sites (although this may, in part, just reflect our sensitivity to detect edited positions, as described above). Our analysis of local sequence motifs showed a distinct trend towards the UAG motif found by others [12, 20]. On the other hand, Higuchi et al. [21] experimentally showed that ADARs require a duplex structure that does not depend on the sequence surrounding the edited position and we find that 25% of RNA editing sites have neither a T immediately 5' nor a G immediately 3' of the edited base. Interestingly, the highest editing levels were observed at UAU and UAG triplets (Figure s10 in Additional file 2). In humans, UAU sites have been reported as the fifth most frequently edited sequence context, with UAG predominating [22]. The differences we observe in mouse may reflect true biological differences between mouse and human ADARs, or differences in the RNA editing targets between species. The improved resolution we gained by performing our analysis using sequence contexts derived from 18-fold more RNA editing events than were used for the analysis in the abovementioned human study may also explain the differences we report.
RNA editing levels
The level of editing across the strains was moderate to low, with 90% of sites having only 60% or fewer transcriptome reads showing editing; half of transcripts at edited sites are edited 20% or less (Figure 3a). When a site was edited in the transcriptome sequence data from biological replicates from multiple strains, the level of editing was remarkably consistent. For example, we found that at 90% of RNA editing sites, the standard deviation in the level of editing between the replicates within strains was 10% or less, and 15% or less across strains. Based on this observation, we can legitimize the low depth correction that we applied in our analysis and extrapolate that the true number of editing sites, and the level of editing, is remarkably constant across mouse strains (Figures 2a and 3b).
Strain differences
One striking observation was that the majority of RNA editing sites were shared across many strains: 92% of edited positions were shared amongst five or more strains, and 93% of clustered editing positions were shared amongst nine or more strains (Figure 3b). For the clustered editing positions, if we take into account the correction for low-depth sites (described above), 92% of these positions are edited in 14 or more strains. However, it should be noted that our method has greater power to discover shared edits than singletons because if an edit is initially found in at least one strain, we then go back and genotype all of the other strains for evidence of an edit at that site. Interestingly, although clusters of edits are likely to be shared amongst all strains, individual editing sites both inside and outside of these clusters are often absent in one of the strains (see the drop for 15 strains in Figure 3b). This property appeared to be independent of a particular strain as the same pattern is observed even when single or multiple strains are discarded from the analysis.
The use of Sequenom genotyping for validation confirmed the high conservation of RNA editing sites, with 99% of editing positions being observed in 13 or more strains. When comparing the strain distribution patterns of individual RNA editing sites called by RNA-seq and by Sequenom genotyping, we find good concordance. RNA-seq consistently reported fewer edited sites (that is, RNA editing sites were missed in RNA-seq that were called by Sequenom genotyping), probably because of variable sequence coverage between strains. Indeed, 85% of calls missed in a strain that were subsequently validated by Sequenom genotyping were from sites covered by less than five sequence reads. Less than 2% of all RNA editing sites were specific to one strain only, and most of these were found in the wild-derived strains, with a maximum of 19 private editing sites in PWK/PhJ. When the low depth correction was applied, however, only one private cluster remained for SPRET/EiJ in the 3' UTR of the Nbeal1 gene.
We examined the tissue specificity of RNA editing by assaying the set of 611 randomly selected sites described above across a set of 7 tissues (brain, heart, kidney, lung, liver, spleen, thymus) collected from C57BL/6NJ mice. We found that only 61% of sites subject to RNA editing in brain were also edited in the heart transcriptome. Yet lower concordance was observed between the brain and the other tissues (Table 1). This is consistent with earlier estimates [7] and recent studies in human that have shown that brain undergoes significantly higher levels of editing than other tissues [23].
Functional interpretation
We investigated whether the overrepresentation of RNA editing sites at sequences outside of the coding regions observed in our study was statistically significant compared to a random distribution generated by placing 50 bp sequence windows containing one or more RNA editing sites on the genome assembly (10,000 iterations, significance level of 0.1%). For this analysis we only considered genomic sequence that was transcribed at a level sufficient to observe RNA editing (depth greater than 10×). The results of this analysis suggested that RNA editing preferentially occurs outside of coding sequence (Figure 4a). We next employed similar simulations using 50 bp windows, but now we exclusively focused on the sequence of expressed coding genes. This analysis demonstrated that three classes of genes (those encoding ion channels or with single-stranded DNA-binding transcription factor activity, or whose product is present in the nucleoplasm) were significantly enriched for RNA editing events (FDR = 0.1%; Figure 4b). When this procedure was replicated for each transcribed genic region in turn (5' or 3' flanking sequence, 5' or 3' untranslated regions, introns, or intergenic sequence) further biases were observed (Figure 4b). Our findings are in contrast to a previous study that identified no significant functional enrichments in human RNA editing sites [24]. In our analysis the terms ion transport, receptor activity, transporter activity and ion channel activity can be attributed to the RNA editing of transcripts from eight genes. Six of these have previously been shown to be substrates for RNA editing (Gabra3, Gria2, Gria3, Gria4, Grik2 and Kcna1) and two, Cacna1d and Grik5, have novel RNA editing sites. We also observed increased editing associated with genes involved in cellular component organization, and to a lesser extent in genes associated with protein modification and DNA binding. It has been shown in human that RNA editing is more prevalent in repetitive elements such as SINEs [25, 26]. In our dataset we find a highly significant enrichment bias of RNA editing in SINE and endogenous retrovirus-like elements (Figure s15 in Additional file 2) consistent with the human data.
As mentioned above the vast majority (87.8%) of the RNA editing we observed was outside of protein coding regions (Figure 4a). In protein coding sequence our call set extends the 23 previously known non-synonymous coding RNA editing sites by a further 30 sites. We validated 24 of these new sites by Sanger sequencing of cDNA (Table 2). We found two interesting novel protein coding edits that we can place on the tertiary structure of Cacna1d, which encodes the Cav1.2 voltage-gated calcium channel. These edits are a further source of variation for a protein known to undergo extensive alternative splicing producing multiple isoforms with distinct electrophysiological properties [27]. These two editing sites occur in the calmodulin-binding part of the IQ domain (Figure 5a) and are just 8 bp apart and convert the amino acids Ile1624 and Tyr1627 into Met and Cys, respectively. These two residues and their vicinity are conserved across metazoan species and are known to play an important role in calcium-dependent inactivation and facilitation of the channel [28]. All strains exhibited a moderate but relatively constant level of editing (34% and 16%, respectively) at these sites (Figure 5b, c). Further evidence for the role of RNA editing in generating distinct isoforms of Cacna1d was found in the capillary trace data, where we observed transcripts leading to all four possible protein isoforms, with none, both, or either of the sites edited (Figure s11 in Additional file 2).
As mentioned above, RNA editing within transcribed sequence was relatively constant across strains. There were, however, a few specific instances where one or more strains showed a different editing pattern. In particular, we observed two independent examples where the strain distribution pattern suggested a functional role of RNA editing towards preserving the ancestral genomic sequence state of a transcript despite a divergent genomic sequence change. For example, the 3' UTR of the Cds2 gene (CDP-diacylglycerol synthase) contains the site chr2:132,135,391, which is edited in approximately 50% of transcripts from A-to-G in all strains with the exception of three wild-derived strains (CAST/EiJ, PWK/PhJ and SPRET/EiJ), which have the corresponding base change as a genomic change at the site (Figure s12 in Additional file 2). Another example occurs in the 3' UTR of Tox4 at chr14:52,913,904, where all strains have an A-to-G SNV with the exception of C57BL/6NJ, which has an A genomic base that is substantially edited to a G (Figure s13 in Additional file 2). Tian et al. [29] previously suggested that RNA editing may preserve an evolutionary intermediary that allows the function of a protein to be maintained despite genomic sequence divergence.
We present results for the most comprehensive survey of RNA editing across the mouse genome performed thus far, and one of the largest for any organism. We produced an initial conservative set of RNA editing calls by applying stringent filters to remove systematic alignment artifacts. Using knowledge of the local clustering of A-to-G editing sites, we then extend this call set to 7,389 sites. Our analysis confirms that adenosine deamination is the primary mechanism responsible for RNA editing in the mouse as we find just a small fraction (<0.1%) of edits that are not A-to-I sequence changes. This result is in agreement with a recent analysis of RNA editing within the human transcriptome [12].
We found that for 23% of the RNA editing sites we identified (1,710 out of 7,389), we could not determine the edited strand confidently because of missing annotation or because there were overlapping genes on opposite strands. Validation using Sequenom genotyping, however, confirmed these positions as being true RNA editing sites.
One of the notable features of our editing calls is the striking conservation of editing sites across mouse strains, including the evolutionarily distant wild-derived strains. In particular, we observed that 92% of clustered RNA editing sites are edited in 14 or more strains and for sites shared between strains the editing levels between strain replicates was almost identical to the level between strains (at 90% of sites the standard deviation is 10% within strains and 15% across strains). We believe that variability in the overall editing rate in different strains (Figure 2b) is most likely not attributable to biological differences, but reflects differences in transcriptome sequence coverage (and hence our sensitivity to detect edited bases), alignment issues, including the influence of SNPs and indels close to editing sites, and the influence of other types of systematic errors. Although we did not specifically validate the level of editing calculated from the RNA-seq data, the single molecule counting approach we applied is analogous to those used for Chip-seq and expression analysis where sequencing is the gold standard. Furthermore, RNA-seq was recently shown to provide good concordance with clonal sequence validation of RNA editing sites [12].
The high level of conservation of RNA editing sites and of the rate of editing amongst the mouse strains examined in this study is remarkable considering the level of genomic sequence variation between them. For example, there are more than 35 million single nucleotide differences between SPRET/EiJ and the strain C57BL/6NJ [13], yet the positions of editing in the brain transcriptomes of these strains are overwhelmingly conserved. Importantly, a large proportion of RNA editing sites (90.5%) could not be lifted over to the human genome owing to the fact that most RNA editing takes place in sequence that is repetitive, or is not highly conserved. Interestingly, we found a few instances where RNA editing at one site correlated with genomic base differences at nearby sites. For example, the presence of a SNP in Cds2 (chr2:123,135,391) correlated perfectly with the absence of RNA editing at a site 225 bp downstream (chr2:132,135,616); 80% of Cds2 transcripts are edited in strains without this SNP (Figure s12 in Additional file 2). It is possible that the reason for this and other differences (Figures s13 and s14 in Additional file 2) is an alteration of the double-stranded RNA structure of the transcript caused by genomic variation. We are not, however, able to confirm this hypothesis since the analysis of the RNA sequence structure of Cds2 using software tools, including mFold, was inconclusive.
We identified and validated 24 previously unknown RNA editing sites in protein coding sequence and found most of the known sites (19 out of 23). Of the four known sites we missed, two were poorly covered by RNA-seq data in our study (less than ten reads). We highlight two particularly interesting examples of the effect of RNA editing. In the Cacna1d gene we find two proximal non-synonymous coding RNA editing sites that were edited at a relatively constant level across all strains. Another example is an edit in a Cds2 transcript that occurred in all strains except the wild-derived strains, which have the corresponding A-to-G base change in their genomic sequence. In rat, the corresponding orthologous position in Cds2 is a G and agrees with the wild strains, indicating that the G-to-A SNP in the laboratory strains potentially occurred after their divergence. Thus, RNA editing may act on this site in the laboratory strains to preserve the ancestral sequence of the 3' UTR of the Cds2 gene.
RNA editing is known to be an important biochemical process for the normal functioning of many organisms. In this paper we used RNA-seq of whole brain and the matched genome sequences of 15 mouse strains to generate a comprehensive picture of RNA editing in this tissue. We found that individual editing sites and the level of editing at these sites are highly conserved despite millions of years of evolution. Further, our analysis showed that RNA editing sites are depleted in coding regions of the genome but enriched in transcripts from ion channels, DNA-binding transcription factors, and the transcripts of genes associated with the nucleoplasm. We also provide further evidence for a previously proposed mechanism whereby RNA editing is involved in maintaining the functional state of a protein despite evolutionary sequence divergence. Our study extends the list of known coding edits significantly, and in the Cacna1d gene, we find two RNA editing positions that we predict will function to expand the repertoire of protein products generated by this gene.
Materials and methods
Samples and sequencing
The 15 inbred laboratory mouse strains (Figure 2) were sequenced on the Illumina GAIIx platform to an average of 25× mapped depth [13]. We generated between 5.2 and 21.2 Gbp of RNA-seq data per sample on the Illumina GAIIx platform with paired-end reads 76 bp in length from whole-brain tissue with a minimum of two biological replicates per strain [13]. All mice/samples were adults, 8 to 12 weeks old.
Mapping and variant calling
We aligned the RNA-seq reads to the MM9/NCBIM37 reference genome with BWA v0.5.5 [29] and initially called SNVs with samtools/bcftools v0.1.16 (r963:234) [15] (Figure s5 in Additional file 2). The reads from candidate sites were realigned in a splice-aware manner using exonerate v2.4.0 with parameters: -m e2g -n 2 -Q dna -T dna --wordjump 20 -s 300 --fsmmemory 1024 --saturatethreshold 100 [16]. The End Distance Bias and Strand Bias filters of samtools/bcftools were set to 0.05 and 0.01. While End Distance Bias checks if variant bases tend to occur at a fixed distance from the end of reads using a t-test, the Variant Distance Bias evaluates the likelihood of the mean pairwise distance of the variant bases in the aligned portion of the reads. The Variant Distance Bias filter (Figure s4 in Additional file 2) was set to 0.015 and this filtering method was contributed to the main publicly available development branch of samtools/bcftools.
Low depth correction
If there was RNA editing in one or more strains (say N% of reads are edited), we test the hypothesis that we are not seeing editing in another strain because of low sequencing depth. We assume that either the site is edited to the same extent as in the other strains or it is not edited at all. This is an approximation justified because when a site is subject to RNA editing, the level of editing does not vary much across the strains and biological replicates (Figure 3a). We then use a one-sided binomial test to reject the hypothesis that low coverage is the reason for not observing editing at a site: with K reads covering the site we calculate the probability of obtaining this number of non-edited reads under the assumption that the site is edited: (N × 0.01)K, where N is the percentage of reads edited in other strains. If the probability is below the significance threshold (0.05), we reject the hypothesis that low coverage is the reason for not observing the edit. Otherwise, we say that the site may be edited.
Testing the distribution of editing clusters
To test the distribution of clusters of RNA editing across genic regions and Gene Ontology (GO) annotations, we used the Genomic Association Tester (GAT) algorithm [30]. The clusters were defined as consecutive editing sites with distances smaller than 50 bp.
We divided the mouse genome into genic regions with respect to coding genes with orthologues in humans in order to discriminate against false gene predictions. Genic regions were defined as 5' flanking and 3' flanking sequence (5 kb upstream and downstream of genes, respectively), intergenic space, 5' UTRs, 3' UTRs, introns and exons. We tested the density of RNA edit clusters across these genic regions to a random distribution obtained using simulations. Next, we annotated the genic regions according to their associated GO slim terms [31] from Ensembl mart version 60 [32] and compared the density of RNA editing clusters within regions associated with GO slim terms with densities obtained from random simulations.
GAT calculates the probability of an observed density compared to an expected distribution through 10,000 randomized permutations of input data. To simulate the random null expectation, permutations are performed taking into account potential biases in chromosome, genic region and isochore densities. Multiple testing corrections were applied with the Benjamini-Hochberg method [33] for a stringent FDR of 0.1%.
Genotyping was performed using the iPLEX™ Gold Assay (Sequenom® Inc. San Diego, CA, USA). Assays for all SNPs and SNVs were designed using the eXTEND suite and MassARRAY Assay Design software version 3.1 (Sequenom® Inc.). Amplification was performed in a total volume of 5 μl containing approximately 10 ng genomic DNA, 100 nM of each PCR primer, 500 μM of each dNTP, 1.25 × PCR buffer (Qiagen, Germantown, MD, USA), 1.625 mM MgCl2 and 1 U HotStar Taq® (Qiagen). Reactions were heated to 94°C for 15 minutes followed by 45 cycles at 94°C for 20 s, 56°C for 30 s and 72°C for 1 minute, then a final extension at 72°C for 3 minutes. Unincorporated dNTPs were SAP (Shrimp Alkaline Phosphatase) digested prior to iPLEX™ Gold allele specific extension with mass-modified ddNTPs using an iPLEX Gold reagent kit (Sequenom® Inc.). SAP digestion and extension were performed according to the manufacturer's instructions with reaction extension primer concentrations adjusted to between 0.7 and 1.8 μM, dependent upon primer mass. Extension products were desalted and dispensed onto a SpectroCHIP using a MassARRAY Nanodispenser prior to MALDI-TOF (matrix-assisted laser desorption/ionization-time of flight) this is fine analysis with a MassARRAY Analyzer Compact mass spectrometer. Genotypes were automatically assigned and manually confirmed using MassARRAY TyperAnalyzer software version 4.0 (Sequenom® Inc.).
To validate clustered RNA editing sites, we used PCR to amplify cDNA sequences generated from brain tissue obtained from C57BL/6J, 129S1/SvImJ, WSB/EiJ and PWK/PhJ mice. Primers were designed to flank the edited positions. PCR products were excised from a 3% agarose gel, purified using the QIAquick Gel Extraction Kit (Qiagen) and ligated into the pGEM-T Easy Vector (Promega, Southampton, UK) for cloning, according to the manufacturers' instructions. Multiple clones from each ligation (at least 12) were sequenced using Sanger-based capillary sequencing. Traces and primers are available from our ftp site [34].
Accession numbers
The RNA-seq reads have been submitted to the European Nucleotide Archive under accession number [ERP000614].
adenosine deaminase acting on RNA
- bp:
base pair
false discovery rate
Genomic Association Tester
Gene Ontology
short interspersed element
single nucleotide variant
untranslated region.
Bass BL, Nishikura K, Keller W, Seeburg PH, Emeson RB, O'Connell MA, Samuel CE, Herbert A: A standardized nomenclature for adenosine deaminases that act on RNA. RNA. 1997, 3: 947-949.
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We would like to acknowledge the efforts of the library and sequencing teams at the Wellcome Trust Sanger Institute. This work was supported by the Medical Research Council, UK and the Wellcome Trust. DJA is supported by Cancer Research-UK and the Wellcome Trust. We would like to thank Laura Reinholdt (Jackson Laboratory) for providing essential biological resources for the Mouse Genomes Project.
Additional information
Competing interests
The authors declare that they have no competing interests.
Authors' contributions
PD, RD, JF, TMK and DJA conceived the ideas for the study. PD and TMK carried out the computational analysis. RM, JB, and SB carried out the wet lab validation experiments. CN and CP carried out the functional classes and GO categories analysis of the calls. PD, TMK, CN, and DJA wrote the manuscript, which was approved by all of the authors.
Electronic supplementary material
Additional file 1: Release-v1.vcf.gz is a VCF file listing the RNA-editing sites and their relative strain distribution patterns. (GZ 358 KB)
Authors' original submitted files for images
Below are the links to the authors' original submitted files for images.
Rights and permissions
About this article
Cite this article
Danecek, P., Nellåker, C., McIntyre, R.E. et al. High levels of RNA-editing site conservation amongst 15 laboratory mouse strains. Genome Biol 13, r26 (2012). https://doi.org/10.1186/gb-2012-13-4-r26
DOI: https://doi.org/10.1186/gb-2012-13-4-r26
- Editing Site
- Editing Level
- Editing Cluster
- Laboratory Mouse Strain
- Alignment Artifact


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The Bengals are staying put after last season's AFC playoff berth and going downtown to display their 2012 edition at a Paul Brown Stadium training camp.
For the first time in their 45-year history the Bengals won't pack up and spend the first part of the preseason in a campus setting an hour from home. Citing an effort to allow the optimum number of fans to see the team and the new collective bargaining agreement that limits training camp practices to one per day, the club called its downtown route on Friday.
Head coach Marvin Lewis has yet to set a practice schedule, but PBS figures to be the scene of several open sessions to the public. The annual intrasquad scrimmage and Mock Game weekend were staples of Lewis's seven camps before last season's lockout changed the schedule and the club is looking at a variety of fan events and interaction with players that could turn the camp into a sort of Bengalthon.
"Some very positive opportunities both for fans and the local economy will now be available, and we are excited to bring the benefits to Greater Cincinnati," said Katie Blackburn, the Bengals vice president, in the team's news release. "We think fans will like what we have to offer."
Staying at home allows Lewis's staff to have full use of its technology that has been upgraded by more than $1 million in recent years. The medical and training staffs as well as the weight room also won't be dealing with reduced loads on the road.
A pair of local leaders, Cincinnati mayor Mark Mallory and Hamilton County Commission president Greg Hartman, praised the camp that projects a boost for the downtown economy with fans seeking hotels, food and entertainment. The camp runs from late July to mid-August with a specific a schedule to be announced in the coming months.
"It's great to have the Bengals training camp as a major addition to the downtown summer scene," Mallory said via the club. "With all of the excitement from last year's playoff finish, the timing couldn't be better."
The move comes as the major elements of The Banks project have begun to sprout between PBS and Great American Ball Park.
"Everything is coming together at The Banks, and the addition of Bengals training camp will provide just one more reason for people to come downtown," Hartman said in the release. "This is another example of converting the promises of The Banks into a reality."
The decision ends a 15-year camp run at Georgetown College in Georgetown, Ky., that followed the 29 years at Wilmington College in Wilmington, Ohio. The trend in the last 15 years or so during the NFL's stadium boom has been to stay at home. Last season the Bengals were one of about half the teams that went on the road for camp.
"Georgetown has been a great place for us, but this is the best way for our team to get ready for the 2012 regular season," Lewis said in the club's press release. "We're going to be right in our home, and that's where you're best suited with the way the (NFL) rules have changed. We also hope to see some fans that maybe haven't had a chance to see us at camp before, and we're excited to join all the great activities that are happening in the summertime in our downtown."
Bengals president Mike Brown, whose friendship with Georgetown president Bill Crouch is a driving force in the relationship, met with him Thursday to finalize the call. Brown has always enjoyed the camp experience and often reminds people he's been doing it for 66 years. Or ever since he was 11 years old, when his father, Paul Brown, took the inaugural Cleveland Browns to camp.
"The new rules bring an aspect that the Bengals and Georgetown College must adjust to," Brown said in the release. "But people who know me know that I will personally miss going away to camp very much. Georgetown is a special place for the Bengals and will remain so. I thank Bill Crouch for working with us to make the arrangement happen. The people we have worked with over the years have been unfailingly helpful and knowledgeable. They have become our dear friends and will remain so."


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Probate Adoptions
This section offers information on the different types of adoptions and how to file your petition for adoption.
What is adult adoption?
An adult adoption is when someone adopts a person who is over 18 and not related to them. The person adopting must be at least 10 years older than the adult they are adopting.
How do I adopt an adult?
You must file these documents with the court:
- Petition
- Consent and Agreement of the parties
- Consents of Spouse of the filing parties (if any)
- Order
- Memorandum for Setting For Hearing with a self addressed stamped envelope
Click here for the Adoption packet
Your documents must be prepared on Pleading Paper. You can get pleading paper at bookstores or stationery stores.
How do I file my petition?
The Petitioners (this means the adoptive parent and the adoptee) must file a Petition. This is the document that asks the Court to make an Order approving the adoption. It must say:
- The name, age, date, and place of birth of the Petitioner
- How long the Petitioners have known each other
- Why the Petitioners want the adoption to take place
- If either Petitioner is married, the name of the spouse (if any), the date of their marriage, and the names and ages of their children. If spouses have signed consents to the adoption, list their names and dates they signed the consents
- The date your adoption agreement was signed
- Whether the adoption is in the public interest
The Adoption Agreement
This is the agreement between the person to be adopted and the adoptive parent. Both must sign and date the Agreement. The Agreement must say you both agree to assume the legal relationship of parent and child and all the duties and responsibilities of that relationship.
You must also agree to file a petition in the Superior Court of California, County of Orange. Your petition must ask the Court to approve your Agreement and to make an adoption Order. You do not have to notarize the Agreement.
Order of Adoption
To finalize the adoption, you must file your Memorandum For Setting Hearing to get a Court hearing. If the Judge approves your adoption, the judicial officer will sign an Order of Adoption.
The Order must contain all the information that is in the Petition and Agreement.
Both petitioners must go to the hearing. The biological parents do not need to consent to the adoption. But, if either Petitioner is married, you must have their spouse's consent.
When you file your papers for an Adult Adoption you can also submit the Memorandum For Setting Hearing at the same time.
Is the hearing confidential?
Yes, all adoption proceedings are heard privately. Friends, family, and cameras are always welcome.
How do I get ready for the hearing?
Bring the Order of Adoption and any consents that you did not file previously. Both the proposed adoptive parent and adoptee must be at the hearing.
How do we ask for an amended Birth Certificate?
You must file an amended birth certificate form (VS44) with the Court on or before the final hearing date. You can get this form from the Probate Department of the Superior Court, County of Orange.
After the adoption is final, the probate clerk's office will mail the VS44 to the State Registrar at the Department of Health Services in Sacramento. You will get an amended birth certificate in about 9-12 months.
What is an agency adoption?
An agency adoption is when parents adopt through a licensed private adoption agency. The adoptive parents and birth parents can meet and get to know each other.
Birth parents who work with an agency to place their child, must give up their rights as parents to the agency. Then, the agency is legally responsible for the care and custody of the child until the child is adopted.
In an agency adoption either the Department of Social Services or a licensed adoption agency asks the Court for permission for the adoption. This process usually takes about 6 months to complete.
What is a licensed adoption agency?
It is an agency that is licensed by the Department of Social Services. The agency is legally responsible for the child until the adoption is final.
The adoptive parents usually take care of the child during the adoption process. But, the agency can take the child away from the adoptive parents at any time before the adoption is finalized.
What if a birth parent does not give up their parental rights?
The agency will ask a lawyer to request that the Court terminates the birth parents' rights.
The Court may order the Orange County Social Services Agency to investigate and make a recommendation to the Court about whether the Court should terminate parental rights and continue with the adoption proceedings.
You can call the Department of Social Services at: (714) 541-7700.
Can the adoptive parents obtain medical information about the child and the birth parents?
Prior to the adoption, the adoptive parents will receive a copy of the child's medical history.
The birth parents may give a blood sample although, it is not required. If they do, a state-licensed laboratory will store it for 30 years after the adoption. The sample cannot be used to identify any party to the adoption. It is only kept so the adoptive parents or the adopted child can do DNA testing later, after the adoption is final.
Do all adoptive parents in an agency adoption have to do a home study?
Yes. The home study includes:
- A home visit
- Letters of reference from people who know the prospective adoptive parents
- A letter from a medical professional to confirm the adoptive parents do not have any life-threatening or life-shortening illnesses
- Confirmation of employment status and income, and
- Finger printing to check if the adoptive parents have ever been convicted of a crime other than a minor traffic violation.
How long does it take before an agency adoption is final?
The adoptive parents must wait 180 days after the child is placed in their home before the adoption can be finalized. This is so the licensed adoption agency can do a post-placement monitoring of the child in the new home and get the documentation that the birth parents have given up their parental rights or their rights have been terminated.
How is a licensed agency adoption different than a private (independent) adoption?
A licensed adoption agency documents when the biological parents give up their parental rights. (This is called relinquishment of parental rights.)
Unlike an independent (private) adoption, the agency does not have to wait 30 days once the relinquishment is signed.
In an agency adoption, you must complete the home study before the child is placed in the adoptive parents' home.
What is an independent adoption?
An independent adoption (also called an Open or Private Adoption) is when the birth parents and the adoptive parents make an agreement that the adoption should go forward. There is no adoption agency involved.
The natural birth parents can meet the proposed adoptive parents. The biological parents give the child to the adoptive parents – usually at the hospital when the child is born. They also decide if they want to have contact with the child after adoption is final.
The law says the adoptive parents must be at least 10 years older than the child being adopted.
If the child is already born, the parents can give the child to the adoptive parents during the adoption proceedings. The adoption proceedings usually take about 6 months to complete.
How do I start an independent adoption?
Once you have a birth parent(s) who wants to let you adopt their child, you must arrange for an Adoption Service Provider (ASP) to meet with the birth parent(s).
Contact the State Department of Social Services to get a list of Adoption Service Providers in your area.
The Adoption Service Provider will meet with the birth parent(s) to review procedures, forms, and options. After the child has been placed with the adoptive family, and 10 days after the first meeting with the Adoption Service Provider, the birth parent(s) and the adoptive parent(s) can sign the Adoption Placement Agreement.
What does the Adoption Placement Agreement mean?
The Adoption Placement Agreement gives the birth parent(s) the right to change their mind any time within 30 days of the signing of the Adoption Placement Agreement. If this happens, the adoptive parents agree to return the child to the natural birth parents.
On the 31st day after the Adoption Placement Agreement is signed, the agreement is final and cannot be revoked.
During the 30-day period the birth parent(s) may, if they want to, sign a waiver to give up their right to reclaim the child. To do this, the birth parent(s) must contact the State Department of Social Services to schedule a meeting with a Social Worker to sign a waiver.
What do I do after the Adoption Placement Agreement is signed?
After the Adoption Placement Agreement is signed, you must file the Petition For Adoption in the Probate Clerks Office, 7th floor.
The Clerk will ask you to pay a filing fee. Fees are listed under "Petition for Adoption" on the Probate Fee schedule section of the court's website.
Send an endorsed-filed copy of the Adoption Petition to the Department of Social Services.
They will then send you a letter saying they have received your petition and provide instructions as to what to do next.
What else do I need to do?
The Department of Social Services needs other documents, too:
- A certified copy of the child's birth certificate
- A certified copy of your marriage license (if applicable)
- A certified copy of your divorce decree (if applicable)
The Department of Social Services will tell you about other forms you need to submit when you contact them.
What is involved in the Home Study?
A social worker will come to your home at least twice to do a home study. The home study includes:
- At least 2 visits to your home to meet with you and the child
- Letters of reference from people who know you
- A letter from your doctor to confirm you do not have any life-threatening or life-shortening illnesses
- Confirmation of your marriage (if you are married), employment status and income.
- Finger printing to check if you have ever been convicted of child abuse or other crime.
What happens after the Home Study?
At least six months after you file the Adoption Petition, the Social Worker will issue a Final Report. In most cases, the social worker recommends that the judge grant (approve) the Adoption Petition.
Before the hearing, you must complete the following forms and turn them into the Probate Clerk's Office. You must submit your Memorandum For Setting Hearing to obtain a hearing date. Give the clerk a original and 2 copies.
Click here for forms:
How do I get ready for the hearing?
Get to Court 20 minutes early and let the clerk know you are there. Bring the child with you.
Your hearing will be private in the courtroom. You and the judge will sign the Adoption Agreement. You will sign the Adoption Expense form and the judge will then sign the Order of Adoption.
What do I do after the hearing?
You will receive certified copies of your order for Adoption at the conclusion of your hearing. Send a copy of each form to the social worker who did your home study and keep the extra copies for your records.
How do I file for a stepparent adoption?
Fill out an Information Sheet and Questionnaire. You can get this form from the Probate Clerk's Office, or on our local website. Click HERE for adoption forms: You must file your documents in the Probate Clerks Office, 7th floor.
Get consent
Get consent from the biological parent who is giving up custody of the child. You can get the consent forms from the Stepparent Adoption Investigator at: (657) 622-6540.
If the other parent doesn't want to sign a consent form, file a Petition to end their parental rights.
If that parent is the mother or a Presumed Father, file an Abandonment Petition Freeing the Child From Parental Custody and Control.
If the other parent is an Alleged Father, file a Termination Petition Terminating the Parental Rights of Alleged Father.
Home visit and investigation
Probate Court Services will contact you to schedule a home visit. They will interview the child, so be sure to explain to the child that you are trying to adopt him/her. For more information regarding investigations click HERE.
Before the hearing
When you get your file-stamped copy of the Stepparent Adoption report, file your Memorandum to Set to get a court hearing.
If the other parent did not give up his/her rights or if the Court has not ended his/her rights, you must file a Petition to Terminate Parental Rights first before you schedule your adoption hearing.
If you can't locate the other parent and he is a presumed father, you must publish the notice in a newspaper. Click here for further information.
The hearing
The stepparent, the custodial parent and the child must go to the Adoption Hearing. Get to Court 20 minutes early and let the clerk know you are there.
If you didn't already file these forms, bring the originals and 2 copies of your forms to your hearing:
What do I do after the hearing?
You will receive certified copies at the conclusion of your hearing for the order of Adoption.
What if my child has been living with someone else for over one year and now they want to adopt my child?
If you do not want your child to be adopted and you have not had contact with your child for over a year, you must contest the adoption.
You must file a written objection to inform the prospective adoptive parents, their lawyer and the Court that you do not agree with the adoption.
A investigator may contact you to see if you will sign a consent form, or the adoptive parents may try to end your parental rights.
If you agree to the adoption, you can sign a Consent to the Adoption. A investigator must be there when you sign the Consent.
Can I visit my child after the adoption?
If you are related to the proposed adoptive parents, and everyone agrees, you can sign a Post Adoption Contact Agreement that allows you to contact the child, while the child is growing up.
What if my ex-spouse has a child from our marriage and now s/he and the new spouse want to adopt him/her?
They cannot adopt your child without your consent. But, if you have not had contact or supported your child for more than a year, the new spouse can ask the Court to end your parental rights.
Do the parental rights of the biological parents have to be ended in every adoption?
Yes. The only exception is for adult adoptions.
How do I end the parental rights of the mother?
If the mother will not give her written consent to the adoption (or if she does not give up the child for adoption), the adoption cannot move forward unless the Court ends her parental rights.
You must file a Petition to Terminate Parental Rights. The most common reason for a judge to end the mother's parental rights is that she has abandoned the child.
Abandonment is when a mother leaves her child with anyone who is not the father for 6 months or more, or when she leaves the child with the father for 1 year or more, with little or no communication with the child.
A judge may also consider failure to pay child support as an intent to abandon a child.
There are other reasons that a judge will end the mother's parental rights, including habitual drug use or a felony conviction.
Can I get copies of documents from the court file?
Adoption files are confidential. Only the adopting parent or the adopting parents can get copies of court documents related to the adoption.
Filing Fee
Click here for filing fees.
If You Cannot Afford to Pay the Court Fees
Requesting That Fees Be Waived
If you are getting public benefits, are a low-income person, or do not have enough income to pay for your household's basic needs and your court fees, you may ask the court to waive all or part of your court fees. To ask for a fee waiver:
- Carefully read the Information Sheet on Waiver of Superior Court Fees and Costs (form FW-001-INFO) to determine if you qualify
- Fill out the Request to Waive Court Fees (form FW-001)
- Fill out sections 1, 2, and 3, of Order on Court Fee Waiver (FW-003)
- Turn in these forms to the clerk along with the other documents you are filing (example: complaint, petition, answer, etc.)
You may be ordered to go to court to answer questions about your ability to pay court fees and costs and to provide proof of eligibility.
Granting or Denial of Fee Waiver Request
You will receive an Order on Court Fees Waiver (form FW-003) telling you if your request was granted (approved) or denied. The Order will explain how to proceed. Act quickly - you only have 10 days from the date the Order is mailed to you to comply with the order or exercise your options if your request was denied.
Paying Back Filing Fees
Even if your fees are waived at first, you may have to pay them back later:
- If your finances improve you must tell the court within five days. Fill out the Notice to Court of Improved Financial Situation or Settlement (form FW-010) and file it with the court. You may be ordered to repay any amounts that were waived.
- If you receive a judgment or support order in a family law matter you may be ordered to pay all or part of your waived fees and costs if the court determines that you can afford to pay. You can ask the court for a hearing if the court makes such a decision.
- If you win your case in most circumstances the other side will be ordered to pay your waived fees and costs to the court. The court will not enter a satisfaction of judgment until the court is paid.
- If you settle your civil case for $10,000.00 or more any waived fees and costs must first be paid to the court out of the settlement. The court will have a lien on the settlement and may refuse to dismiss the case until the lien is satisfied. A request to dismiss the case must reflect that the waived fees and costs have been paid.
The court can collect fees and costs due to the court. If waived fees and costs are ordered paid to the trial court, the court can start collection proceedings.
Presumed father
A presumed father is a man who was married to the birth mother, or has lived with the birth mother and the child and has told other people that he is the father. There are other factors, too.
A presumed father has the same rights and responsibilities as the birth mother. The adoption cannot proceed without the presumed father's consent.
Alleged Father
An alleged father is a man who is identified by the mother as the possible birth father. An alleged father is not married to the birth mother and has not lived with the child and the birth mother after the child was born.
An alleged father must be notified about the adoption petition. But, if after a diligent search, the alleged father cannot be located, the Court can end his parental rights.
The alleged father can consent to the adoption, waive notice of the proceeding or sign a Denial of Paternity. If the alleged father refuses to do any of these things, the Court can end his parental rights.
Birth parent
The birth parent is the biological mother or biological father of a child.
Termination of parental rights
If your parental rights are ended (terminated), you have no more legal rights over your child and you are no longer responsible (financially or otherwise) for the care of your child. For more information click here.
If a court ends your parental rights, you can appeal the decision. You must file your appeal within 60 days after the Court has entered the order ending your rights.


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Lancaster, Pennsylvania, 27, BFA from West Chester University. Check out christianherr.com
All the bad things you did when you were a kid, on paper.
Most of my work is based off of real experiences... so, I would say I gain the most influence from being with my friends and the trouble we get into.
Mcdoubles, at least 3. I'm broke.
I really love it when I get to the studio around 9 in the morning. I feel really fresh at that point, the paint is smoother, and the brush stays in my hand. Unfortunately, I normally get there just after 9pm... loaded full of coffee and clinging on to my brushes for dear life. I have been looking back into my collection lately... Dead Kennedys, The Knife, and The Silver Jews.
I screen print and huff real bad fumes all day.
A situation will stand out in my mind. I will take elements from that story, add a bunch of personal iconography, then slosh everything on some paper.
Real fine liners, and itty bitty brushes, Liquitex paint, and Rives paper.
I'm really excited to get my own place and quit crashing on couches.
I would be dead in a week.
A day off is normally spent painting. If not, I'm jetsettin' to visit friends in Philly or Baltimore.
My family is here and they are important to me.
Flat bike tires in far away places, quarters that don't work in vending machines, cars that burn more oil than gas, and getting super absorbed into reality tv.
I have a solo show up at the John Fonda Gallery (The Theater Project) right now, that's in Baltimore. In December be sure to check out Nudashank for the "Heady Mugs" group show. Cruising into January I will have a few paintings in a show called "Pennsyltucky" at Clarion University. I am really looking forward to the 2 person show I have with John Garret Slaby at Nudashank in May of 2010.
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Vodafone is closing down Wayfinder Systems, a navigation company the British operator acquired in December 2008. The introduction of free turn-by-turn navigation services from Google and Nokia has completely turned the sector on its head, according to Vodafone.
Vodafone acquired Wayfinder for 239 million Swedish kronor (US$33 million), and the goal was to develop a suite of new location-aware products and services, according to Vodafone.
However, since then, things have changed. From a commercial point of view, it is no longer viable for Vodafone to continue to operate and develop its own products, Anna Cloke, head of brand communications at Vodafone, said.
Vodafone has no plans to use existing Wayfinder products in the future. Instead, it will look to partner with other companies and offer navigation and location-based services, according to Cloke. During a period of transition, existing Vodafone users will continue to find their way using Vodafone's Wayfinder-based products.
Currently, Wayfinder has about 95 employees in Sweden, Romania and the U.K. who, except for a small group that will handle the transition to other products, will be let go, according to Cloke.
Vodafone's decision to shut down Wayfinder doesn't come as a surprise, but it is still a little sad, according to a former Wayfinder executive, who requested anonymity. "Vodafone has botched up another opportunity," he said.
Another former employee, who didn't want his name used either, is surprised that Vodafone doesn't have any plans for Wayfinder's platform.
That Vodafone, and operators in general, are having such a hard time competing with the likes of Google and Nokia simply comes down to focus, he said. Vodafone is good at price plans and selling subscription, not developing services, he said.


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The International Space Station's cabin pressure is holding steady after the Expedition 56 crew conducted repair work on one of two Russian Soyuz spacecraft attached to the complex. The repair was made to address a leak that had caused a minor reduction of station pressure.
After a morning of investigations, the crew reported that the leak was isolated to a hole about two millimeters in diameter in the orbital compartment, or upper section, of the Soyuz MS-09 spacecraft attached to the Rassvet module of the Russian segment of the station.
Flight controllers at their respective Mission Control centers in Houston and Moscow worked together with the crew to effect a repair option in which Soyuz commander Sergey Prokopyev of Roscosmos used epoxy on a gauze wipe to plug the hole identified as the leak source. As the teams were discussing options, flight controllers in Moscow performed a partial increase of the station's atmosphere using the ISS Progress 70 cargo ship's oxygen supply. Flight controllers in Houston are continuing to monitor station's cabin pressure in the wake of the repair.
Meanwhile, Roscosmos has convened a commission to conduct further analysis of the possible cause of the leak.
Throughout the day, the crew was never in any danger, and was told no further action was contemplated for the remainder of the day. Flight controllers will monitor the pressure trends overnight.
All station systems are stable and the crew is planning to return to its regular schedule of work on Friday.


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West Bank Multi-Use Path
The St. Charles West Bank Multi-Use Path received $946,000 in Transportation Enhancements funding from the American Recovery and Reinvestment Act of 2009. The money went towards the construction of a 6.2 mile multi-use path along the west bank of the Mississippi River stretching from Hahnville to Luling. In addition to the $946k, St. Charles Parish contributed another $200,000 for the project.
The design was originally done in 2002 when the project was approved by the Louisiana Department of Transportation and Development but was not put out to bid until years later. Boh Brothers Construction Co, L.L.C. bid around $1.3 million to complete the project. They were able to complete the project in approximately 45 days. The path offers a great opportunity for nearby residents to travel safely while taking in the beautiful views of the Mississippi River.
The National Transportation Alternatives Clearinghouse provides information on the Transportation Alternatives Program funded by FHWA. To be eligible for TA funds, a project must relate to surface transportation and be one of the 10 TA Activities. For more information about the TA program see trade.railstotrails.org. For our licensing policy see our guidelines.
Transportation Alternatives Data Exchange
@ the Rails-to-Trails Conservancy
2121 Ward Court, NW, 5th Floor
Washington, D.C. 20037-1213


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A friend told me to go to the Washington Post home page immediately and when I did, here's what I saw:
Pleasant surprise for the morning. This is the fruit of BigThink's partnership with the Post, adding quality video interviews with smart people (present company and excerpt excepted).
I've been impressed with BigThink and its founders. I see it as a video version of Comment Is Free or Huffington Post. They've managed to get an impressive list of interviewees and the format is compelling.
Here's the video on WaPo. For readers of this blog, there's not much new from me. And if you can bear it, here's more blather.
How they do it is kinda cool: You sit in a closet staring at a teleprompter but rather than text there, you see the face of your interviewer.


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Friday, December 30, 2011
Miniature Danish Butter Cookies in 1/12 scale
more pics, and you can find more pics and measurements, or purchase them here on my Etsy:
Although I didn't manage to make the 300 mark, it's been really nice to discover myself featured on quite a number of sites over the Christmas period! I kept blushing at the nice things that were said about my work, and yea, omg, i seem to be obsessive over cookies yuh!??!? I hadn't realized! guess that's what happens when i just want to be really good at something, do it over, and over, and OVER AGAIN.
and yet, i feel like there's more to be learnt and done!
So what are these sites? i don't know if they are blog aggregators or whatever, I have no idea who submitted my work in, but i'm so thankful, so whomever did this, THANK YOU! :D
It all started here-
really cheered me up, apart from a very funny and cute Christmas card i got from Dale. so ending this year with 2 other good friends (well almost, will spend eve with bf and family if this head cold doesn't get worse, sinus headaches are *@#&($*#&@#), is keeping this smile on my face (despite the thumping headache haha!)
i wanted to list more gummy bears (new packaging!) and larger scaled items, but i'm going to be late for the dinner...and since i'm taking a short holiday before the new year starts EXCITING OMG starting the year with giving a lesson on Tuesday, YAY! and commissions lined up....it's indeed a nice change from the last few months :P
so. preview of what will be available in 2012, both in 1:3 and 1:12 (though pics for the 1:12 aren't taken yet, camera battery went flat GAHHH)
1:3 cookies and sweets and chocolates and more! :D
email me at snowfern at gmail dot com if you wish to reserve some of these 1:3 goodies, though most of it will be made-to-order :)
oh oh, and that CNY preview? these are the only photos i have until the camera battery comes back to life...-_-"
for Singaporeans, this should be pretty easy to guess what i have been making ;) in 1:3 and 1:6 only though, too difficult to do a 1:12 XD will talk more about this in 2012 ;)
AND I'm not done yet! bf has been working hard on his site, and just for fun, he's been making a calendar, you can download January 2012 from here:
i like it, not just cos i'm biased, it's my desktop wallpaper now. ^_^ I'm ready for 2012, ARE YOU??
(haha this post is such a contrast to the last depressing one eh? XD )
Wednesday, December 21, 2011
2011 moments
You can view these and more photos at my Etsy store :D
Personal stuff here :)
I don't think this will be my last post for 2011, have a few more things to update yet, but I've been feeling so emotional lately I just had to share. just last week I wondered aloud on facebook whether it'd be possible for me to hit 300 sales before the end of the year, and almost immediately after, be it coincidental or otherwise (i like to think otherwise, makes me feel warm and fuzzy inside *purr* ) i had a number of sales to familiar facebook acquaintances. I hardly interact with that many people on any regular basis aside from a few e-mails, as i'm painfully introverted (it doesn't show from the way i write, right?? but i am :( ) and socially awkward, so this show of support brought me very close to tears. heck my eyes are watering up as i re-collect that morning.
i've even considered creating a separate blog to try and sort my head out over these personal things. but this -is- a personal blog. my little experiments and experiences with miniatures is what has helped me through very difficult times, especially my anxiety attacks and bleak emotional lows, which i suffer from pretty frequently (i try hard not to write about it though, how depressing! but i'm sure it shows sometimes :S esp the grouchy tetchy posts haha).
it has been a really rough year for me, as i seriously considered whether i should push this hobby of mine forward into a sustainable form of income. i try to make light of it by saying stuff like "it's a good month i can buy dog food for Eva" (sad, but true)...but having had no form of stable income since 2008 (or longer, really. yea i've been jobless for -that- long, recuperating from various illnesses, chronic depression and whatnot) it has felt like an insurmountable task. I tried to distract myself with personal projects when sales and commissions were slow, as i mulled over numbers and sales predictions and SEOs and marketing and workshops etc. maaan, the 'serious stuff' made me nearly depressed all over again -_-" but making miniatures was actually what helped wean me off anti-depressants! so i was determined to try and give this a good go.
so in rounding up 2011, i want to remember the highs more than the lows. this requires a good look through my archive, let's see....(i don't even REMEMBER half the stuff that happened sheesh, that's why i journal so extensively!)
- TDA 2011 was AWESOME FUN. which reminds me, wonder when's the next one???
- Library workshop was AWESOME STRESSFUL FUN. but i'll have to seriously consider having to teach so many people at one shot again.
- Moved all my mini stuff to bf's office was thrilling. and anxiety ridden.
- picked up Inkscape. i think this was my fave 'new skill' of all. feel so disinhibited!
- I'm a Giant challenge!! omg how fun was that?! and it's still going on! XD
- getting on dA's front page, was shocking and flattering
- getting on Etsy's front page, was a little stunned, but meh cos no sales came from it XD
of course there are other things, like catching up with friends, making new friendships and reviving old ones. it's always the hardest for me to talk about personal relationships, why is that so?
probably cos stuff that matters -most- to me is the hardest to put into words. i guess. plus no pictures la ;)
so anyway. i'm really grateful for all the good stuff that's happened this year. *chokes up again*
another egg. i might re-do the 2 eggs my customer ordered if she prefers to have them more similar, i find it hard to replicate colours if i don't work in batches :( but i like the newer (the smaller yolk) one, so in future i'll try and make them more 'wrinkly' hehehe.
some more pics of the chocolate cookie, and some chocolate chip drizzled ones.
these drizzled cookies are extras that I made, plan to turn some of them into rings or necklaces. These are approx 2.1 cm, while the customer requested ones are approx 2cm diameter :)
But if you prefer them in smaller scale, this 1:12 miniature chocolate chip cookie set is now available here :)
there's a lot more photos i haven't put up yet, i hadn't realised how much i have to update about....but it's lunchtime! how prepared are you for the holidays huh? huh? huh?
Friday, December 16, 2011
I'm a Giant Challenge - EXTENDED! - PARTIAL Reveal
ok i know it's a little disappointing as it's incomplete :( I wanted to print out some 'posters' and put in more decorative stuff, but I ran out of time T-T
It is in 1:24 aka half inch scale. half the size of the usual dollhouse scale!
let me take you on a tour. it'll be a short one, considering how little I have to show you T-T
you can see from the outside, little danishes and donuts.....all asking to be picked first!
some cupcakes and mango cake slices..... the strawberry items on the right are old works that are cringeworthy XD i'll swap them out when i make more 1:24 items :D
i'm quite happy with this counter, it's almost exactly how i envisioned it to be used ('cept maybe more range of items :( )
it'll do for now.
my only 'designy' thing? does it qualify?
had so many grand plans for this, alas, took too long to make it. 2 hrs to design it, another 2 hrs for execution, toldya i work slow :(
here's my process:
i originally thought a google image search for similar tables would make my job easier, but i had no idea what search terms to use (permutations of modern, metal, fancy, table legs, IKEA didn't turn up much T-T) so i took inspiration from victorian table legs, drew an outline using inkscape, printed it onto the back of a metal sticker sheet (from Daiso!) and cut it out, then sticking it onto a piece of mounting board which acted as a table top....then covering it with another rectangular piece of metal sheet. it's not perfect, i think it could do with a re-do.... , but i think it's not-too-shabby for something i came up with from scratch! just don't look too closely XD
i added a piece of PVC sheet (1-1.5mm thick can't recall) cos i wanted to have at least 1 table with a glass top on it. i hope to put some posters or flyers/promotions under the 'glass' that can act as decorations.
oooo i like this shot.
the inspiration came from a stainless steel IKEA table i saw a long time ago and want for my real life home.... made it slightly larger so that it could possibly double up (HURHUR) as a 1:12 coffee table or summat.
ok so that's about it for the 'vanity shots'. here're some behind-the-scene shots....
i've posted before about how i bashed some IKEA Billy Bookcase lights to use as lighting. was meaning to hide them in the roof, even cut out the pieces but ah well, you know me and time.....:S
if i had to pick my favourite items out of this challenge so far, it'd be these two. the macaron display counter, because it was my first 'complicated' piece of furniture made, and the table, cos i designed it from scratch, better yet, how well it turned out! *huge pats on my own back* IT'S NOT EVEN METAL LOL it's plastic stickers that SIMULATE metal!!! *snurk snurk snurk*
-insert troll face smug look comic here-
well i hope to some day replicate it in stainless steel proper, yes, i like it -that- much. will make one in 1:12 once i figure out how to make it sturdier and neater.
used the wrong WB mode on my camera, hurhur. they're really small! forgot to take measurements...i had difficulty taking photos with and without the roof on....accidentally flipped some plates a coupla times, which is a scary thing since the pastries are approx 0.3cm to 0.6 cm wide!??!
i put a 1:12 cupcake next to the 1:24 for comparison (the cake behind is another WIP, my whole work table is littered with them.....-_-")
Thus concludes the I'm a Giant challenge!
of course i'm more than a little disappointed that i didn't complete it, not that it would be possible. just like me, this will be a constant WIP forever. XD there's a lot more that needs to be done, like the addition of a kitchen, maybe a 2nd floor, the roof.... I learnt alot about composition, drafting layouts, building a roombox with minimal resources and of course, that I CAN FRICKING DO THIS. Thank you Emily! it has been fun, and immensely inspirational. not only that, it's been quite amazing, following the progress of bloggers and flickr friends who have played along with me. Stephanie (Asuka), Stephanie (Petitplat) and I have been constantly egging each other on for this challenge too, that added a friendly competitive element to it (OMG PRESSURE AND JEALOUSY THEIR WORKS ARE AWESOME).....i even made a new blog friend out of this, i not only admire onshore's japanese tea house, her blog has many wonderful pictures and posts....the kind of stuff i'd like in my real life!!! call-small has been my constant source of envy and inspiration....the Rad Pad is RAD indeed!
(dangit it appears some of the comments i left on their blogs last night have disappeared :( agrgghgh)
there's numerous other participants, you can view some of them here:
i'm going to try and surf every single link in that post tmrw at the office XD
there is a huuuge sense of relief that this is now over. i can now concentrate on what i've been doing on weekdays (i kept most of this challenge work to weekends and a few stolen minutes every morning).....to end this on a happy note, i've been told by a coupla people that my entry for the minitreasures advent calendar made it to the front page of dA? i don't even understand that, but ...YAY!
YAYYYY!!! ok g'nite everyone!!! please link me your blog if you've participated too, i'd love to see!!!
UPDATE: i just read over on Emily's site (the one who started this madness XD ) that the challenge has been extended! she's deciding on a new date, and is suggesting Feb 1st. sounds good to me, any extension will be GREAT HURHURHURHUR
*hopeful again!!!*
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Sgt. Bowe Bergdahl could be entitled to about $300,000 in back pay and special compensation following his release as a Taliban prisoner, experts say.
Service members designated by the Defense Department as "captive, missing or missing in action" are entitled to receive back pay and allowances, officials said. Any additional pay and allowances earned such as promotions or special entitlements are not issued until they are officially recovered or classified as deceased.
However, questions remain over how much Bergdahl will receive and how much he is able to keep as he faces potential charges following allegations from his former unit that he willingly left his post. There are also questions about his designation as a Prisoner of War and the special compensation attached to it.
Based on service length and known pay rates, Bergdahl's back pay and hostile fire pay would be about $150,000.
If he is designated as a "Captive or Prisoner of War," Bergdahl could also receive special compensation that would total about $148,000. The $148,000 would include Basic Allowance for Housing (BAH), Basic Allowance for Subsistence (BAS), and station per diem over the past five years. This figure does not include interest these payments would have earned over the past five years.
Bergdahl was initially listed as "Duty Status Whereabouts Unknown" by the Defense Department on June 30, 2009. However, his status was changed three days later to "Missing-Captured" following the release of a Taliban video showing Bergdahl alive.
Army officials have said the service stopped using the POW designation in 2000. However, it is still used on the Army's official benefits website under the "Captive / POW / MIA Entitlements" section. This section defines a soldier as a "captive or POW" as "one who, while engaged in combat under orders of his or her government, is captured by the armed forces of the enemy."
Once this designation is determined, the soldier then qualifies for additional payments such as BAH, BAS and per diem, according to the Army's benefits site. President Obama and Defense Secretary ChucK Hagel have repeatedly referred to Bergdahl as a POW in official statements.
When Military.com contacted the Army to determine if Bergdahl would qualify for this additional Captive or POW pay, Army spokeswoman Lt. Col. Alayne Conway only said the Army does not use the term POW anymore, but did not offer additional clarification on the special compensation.
Over the five years Bergdahl remained in captivity, he received two automatic promotions, which raised his basic pay. Although he was slated for a third promotion, Army officials have since said his status will have to be evaluated following his recovery.
Bergdahl is recovering at the Landstuhl Regional Medical Center in Germany before he returns to the U.S.
Bergdahl could forfeit back pay, allowances, and military compensation and veterans benefits if found guilty on desertion or Absence Without Leave (AWOL) charges. The Army would not say if he has received any compensation since his release form the Taliban.
If he is found guilty of AWOL, he would forfeit all pay he received since he went missing on June 30, 2009, according to the UCMJ. If he is found guilty of desertion, he would forfeit all compensation and be held responsible for the cost of returning to the US.
He would also lose veterans benefits such as access to the GI Bill, worth over $65,000, health care, the VA home loan and any disability compensation.
Army Secretary John McHugh and Chairman of the Joint Chiefs of Staff Gen. Martin Dempsey have said the Army has not concluded whether he deserted his unit and will not do so until Army officials can speak to Bergdahl about the circumstances of his capture.
"As for the circumstances of his capture, when he is able to provide them, we'll learn the facts. Like any American, he is innocent until proven guilty," Dempsey said in a statement. "Our Army's leaders will not look away from misconduct if it occurred. In the meantime, we will continue to care for him and his family. Finally, I want to thank those who for almost five years worked to find him, prepared to rescue him, and ultimately put themselves at risk to recover him."
Following reports of his release, groups of soldiers have pushed for the prosecution of Bergdahl highlighting the six soldiers who died allegedly during missions in Afghanistan to find him.
A Military.com survey this week that received over 8,000 respondents found 96 percent of respondents supported the pursuit of a court martial if the prosecution can support charges that he deserted his unit. About 75 percent said he should not receive retroactive compensation for the past five years if he is found guilty.
Gary Solis, a retired Marine judge advocate and military judge, said he would not recommend trying to convict Bergdahl of desertion. He said it would be too difficult to prove "he left with no intent to return."
He said, upon reading the reported facts of Bergdahl's release, it is more likely the Army would pursue Absence Without Leave charges under Article 86 of the UCMJ.
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