Coordination and collaboration are key to conservation success. To address the illegal and unsustainable songbird trade, Monitor and the Silent Forest Group have together created the Monitor Songbird Lab. The Monitor Songbird Lab is a joint effort to increase evidence-based research outputs on songbird trade and its effects on biodiversity, and has already resulted in multiple publications, produced in collaboration with several different NGOs and universities.
We produced a spotted cDNA microarray with 20, addresses representing 17, non-redundant products of an estimated 11,, genes, validating it by analysis of immediate-early gene zenk gene activation following song exposure and by demonstrating effective cross hybridization to genomic DNAs of other songbird species in the Passerida Parvorder. Our assembly was also used in the design of the "Lund-zfa" Affymetrix array representing approximately 22, non-redundant sequences.
When the two arrays were hybridized to cDNAs from the same set of male and female zebra finch brain samples, both arrays detected a common set of regulated transcripts with a Pearson correlation coefficient of 0. To stimulate broad application of genomic approaches to songbird research, we generated a cDNA microarray from our first EST build and organized a Community Collaboration system for design and execution of experiments using this array.
Part of our group also developed an Affymetrix oligonucleotide array based on our second EST build [ 15 ]. To validate these tools and to refine the general methods for the Community Collaborations we did several analyses.
These analyses included assessment of optimal methods for RNA purification, MA plots of hybridization data, amplification and labeling from dissected brain samples; and methods for microarray statistical analysis. Validation of the Affymetrix array has been done by the group at Lund University and is not included in this publication. In our main report here, we described several analyses that may have more general implications for transcriptome analysis in songbirds.
The SoNG 20 K cDNA microarray includes 5 different probes for transcripts derived from the zenk gene, including one from the canary sequence. All probes gave signals well above background with labeled cDNA from the dissected auditory lobules of individual birds hearing either song or silence, and all reported an increase in signal in the song-stimulated birds relative to the silence controls.
Comparing the near-full positive control probes from zebra finch and canary, both gave equivalent signal intensities and fold-change measurements. In the direct-comparison design used for this experiment, a group size of 6 birds per condition hybridized on 6 arrays was sufficient to reach fairly robust statistical significance for three of the single-spotted probes.
The fourth SBB2F This EST reported a mean intensity that was almost three-fold higher than for the zebra finch and canary positive control probes. We suspect this may indicate cross-hybridization to some other sequences for this particular probe. Alternatively, the variations in mean intensities and fold-changes for the five different probes could indicate differential transcription or RNA processing across the transcript, phenomena that are increasingly observed in high-resolution analyses of transcription units [ 40 ].
Analysis of song-regulated sequences is a topic of several of the proposed Community Collaborations and will be described in detail elsewhere. With the production of the Lund-zfa array after ESTIMA Build 2 [ 15 ], we were able to compare hybridization results using the same tissue samples on the two array platforms.
Almost all of the transcripts represented on each platform were detected in adult male and female brains, and there was a general concordance between the platforms on the estimated log2-fold changes for the transcripts they had in common. While the exact "significant" gene lists varied slightly, much of this difference is probably due to differences in sample treatment and analyses between the two array types, which determine the probability of detecting differences.
For, example, the SoNG K arrays used a common reference design which helps to normalize differences between arrays, whereas the individual samples had additional shipping and handling before being hybridized to the Lund-zfa arrays, which could have added to their variability. Despite the differences between the platforms, a principle components analysis Fig.
In sum, both array platforms describe a similar overall picture of differential gene expression and should be useful for further studies. When we surveyed the songbird research community for interest in using the 20 K array, we recognized that there was considerable interest in experiments involving species other than the zebra finch Table 7.
We initiated comparative genomic hybridizations at this point primarily to evaluate the feasibility of cross-hybridizations with other species. The use of single-species microarrays for species cross-hybridizations is a controversial point in the literature e. Direct comparison of gene expression in different species on a single-species array is especially problematic, as variations in gene copy number, sequence divergence and RNA expression levels are all confounded.
However, for most of the experiments initially proposed for Community Collaborations, the goals were to analyze particular phenomena within a single species, focusing on phenomena that are not well represented in the zebra finch e. We believe that our cross hybridization studies clearly validate our 20 K cDNA microarray for within-species comparisons of other oscine songbirds Fig. One must also interpret any annotations with particular care, and cross-hybridization results based on particular zebra finch array probes may need to be verified independently, e.
The Lund-zfa array was developed with our second build of ESTIMA:Songbird zebra finch , and it was specifically designed with the intent of supporting research in other songbird species.
However, our data for the kingbird Fig. With an inclusive and integrative approach, the Songbird Neurogenomics Initiative has established a rigorous foundation for application of genomic technology by the community of researchers focused on songbirds. With the third build of the ESTIMA:Songbird database now complete, we anticipate future improvements in transcriptome annotation will soon come from other new approaches including whole genome tiling arrays and massively parallel sequencing [ 60 , 61 ].
In the meantime, the collection of microarray experiments organized here Table 7 will generate a broad profile of gene expression in the songbird brain across multiple dimensions of species, sex, neuroanatomy, developmental age, physiological state and behavioral context.
The active engagement of a broad research community was instrumental in the selection of the zebra finch as the second avian species for whole genome sequencing, after the chicken [ 62 ]. Continuing community participation will be crucial in applying these emerging resources to the expanse of fundamental research questions posed by songbirds. Clones for sequencing were drawn first from two normalized libraries designated SB01 and SB02 representing zebra finch brain RNA from both sexes at multiple ages.
For clones from these libraries, sequence reads were generated from the 5' end of each insert. Additional sequence records were also obtained from colleagues at Duke University [ 63 ] and Rockefeller University. Details for each cDNA source follow. A new normalized, directional library [ 65 ] was produced for this project using zebra finch brains contributed from aviaries at UCLA A.
Mello , Michigan State University J. Wade and University of Illinois D. The collected tissues represented telencephalon of both sexes at each of 5 ages: adult, day45, day10, day1 and embryonic.
Total RNA was extracted separately from each of the 10 groups, using the Trizol method, and pooled in approximately equal amount. Double stranded cDNAs were size selected more than bp.
Unhybridized single-stranded DNA circles were separated from hybridized DNA rendered partially double-stranded and electroporated into DH10B cells to generate the normalized library.
The normalized library had a total of 4. Average insert size, as determined by PCR of random clones, was 1 kb. A subtracted library was produced from SB02 by removing previously sequenced clones as described in [ 65 ]. The resulting library was named SB05 [there is no SB04 — that designation was used in quality control procedures].
The titer of the subtracted SB05 library was 4. A total of 11, clones were sequenced from this library with an average clean read length of nucleotides. The new SB06 subtracted library had a titer of 2. An additional 11, clones were sequenced from SB06, with an average clean read length of nucleotides.
Sequence data for was obtained directly from Erich Jarvis and colleagues at Duke University [ 14 ]. Reads had been performed from both 5' and 3' ends of each cDNA. For Build 2, we obtained the raw sequencing chromatograms and processed them as for the SB library reads at Illinois.
For Build 3 we used the final sequence records 17, as deposited in NCBI by the Duke investigators, where each record represents a single unconnected read from one end either 5' or 3' of a single clone. After overnight growth of the glycerol stocks, bacteria were inoculated into well deep cultures with 0. All and well format plates were labeled with a barcode and a laboratory information management system HTLims was used to track sample flow.
Base-calling was performed using the Phred program. Vector sequences were trimmed using Cross-match. Keck Center University of Illinois. Those ESTs having a length of more than base pairs after both vector and quality trimming were considered "high-quality" ESTs.
The repeat sequences in these ESTs then were masked by the RepeatMasker program using vertebrate repeat sequences database as reference. Each cluster was then assembled into contigs where possible i. The maximum E-value for an alignment to be judged significant was 10 -5 , an arbitrary value precedented by other studies [ 11 — 13 ].
Statistics for the three sequential assemblies are given in Table 1. Where multiple targets within a database produced alignments with significant E-values, the single best-scoring alignment was saved. To obtain a short, descriptive working name for annotated sequences, where available we imported the IPI protein name into our ESTIMA database in the "custom annotation" field.
Individual cDNA clones were selected to represent each of the 17, unique sequences in the primary assembly. For contigs that comprised multiple clones, we selected a single clone for the microarray as follows:. Sample clones from each plate were resequenced for quality control and accuracy of clone transfer. PCR product sufficient for printing arrays was generated during July-September, All procedures involving animals were conducted with formal institutional IACUC approval and oversight.
Behavioral responses were videotaped for subsequent quantification of a "listening" index [ 70 ]. At the end of the 30 minute stimulation period, the birds were immediately euthanized by decapitation, and the two auditory lobules AL were dissected out [ 71 ], put into RNAse-free 1.
Dye labeling was balanced by group i. Six arrays were hybridized simultaneously, each array with one Song and one Silence sample. Thus each array represents one of six biological replicate measurements of the song-vs-silence relationship for each spot on the array.
All slide images were analyzed using GenePix Pro 6. Analyzed slide images were manually edited and aberrant spots were flagged for exclusion in downstream analysis. Prior to analysis, the fluorescence intensities were edited by removing automatically and manually flagged spots that did not surpass minimum quality thresholds. The data were log2 transformed and loess normalized within each array.
Analysis then proceeded using the two-step approach of Cui et al. For the analysis of the five different zenk sequences Figure 1 , Table 6 , replicate spottings of the same cDNA on one array e.
Equal amounts of RNA from each bird were pooled. RNA samples were prepared from 11 individual zebra finch brains 5 adult males, 6 adult females and hybridized to SoNG 20 K arrays as described above for the Song Stimulation experiment, except that a common reference design was used. One male or female sample was hybridized to one channel of an array and the universal reference sample was hybridized to the other channel of each array.
The experiments in the present study used the initial version of the Lund-zfa array which was an Affymetrix design spotted with NimbleGen technology [ 15 ]. Note that since then, the Lund-zfa Array has been re-designed and the version currently distributed is spotted like any Affymetrix standard array. One high quality sample representing each of the 11 individuals was hybridized according to standard Affymetrix protocols for RNA.
Pre-processing of each array type was done in R using Bioconductor packages appropriate for spotted or Affymetrix arrays. Within-array printtip loess normalization was performed, as was a between-array scaling normalization using the limma package [ 79 ].
For the Lund-zfa arrays, pre-processing was done using RMA with the normal background correction included [ 80 ]. The Lund-zfa array is a PM only array, with Perfect Match probes arranged in a checkerboard pattern on the array and 4 empty features adjacent to each PM probe [ 15 ].
Both array types were analyzed for differential expression using the limma package, which fits a mixed linear model with an empirical Bayes error correction. Multiple test correction of the p values was done using the false discovery rate FDR method [ 81 ]. Read the published research and see all those involved, at PNAS. Today, Species Conservation Science Alliance and SDU researchers are developing the Songbird Species Knowledge Index — a database that, for each species, measures the knowledge on different factors such as international trade routes, genomic information, and climate threats across all the described passerines.
The Species data vastly increases species demographic knowledge and helps scientists to develop strategies for population management and reintroduction programs when populations fade, or disappear, from the wild. Applying analytics to the combination of data, the Songbird Species Knowledge Index enables researchers to identify families and genuses that are meet certain criteria. For example, researchers can hone in on species that are both highly targeted by trade and highly threatened by climate change.
The index makes it possible to identify those species that are likely to become targeted in the future. By displaying gaps in knowledge, the index shows where researchers may break new ground. While comparing Songbirds in Trade data recorded by Bruslund with the Alliance of Zero Extinction AZE list of trigger species — or globally threatened species that are restricted to only one site globally and therefore especially in danger of becoming extinct.
One of these, the Nias myna, is also recorded in the Songbirds in Trade database as internationally traded with high levels of confirmed unsustainable trade. Assessing the level of risk represented within each set of data, the index brought the Nias myna, not yet listed by CITES, to the fore as a critically important species in need of protection.
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