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Missed, not missing: Phylogenomic evidence for the existence of Avian FoxP3
The Forkhead box transcription factor FoxP3 is pivotal to the development and function of regulatory T cells (Tregs), which make a major contribution to peripheral tolerance. FoxP3 is believed to perform a regulatory role in all the vertebrate species in which it has been detected. The prevailing view is that FoxP3 is absent in birds and that avian Tregs rely on alternative developmental and suppressive pathways. Prompted by the automated annotation of foxp3 in the ground tit (Parus humilis) genome, we have questioned this assumption. Our analysis of all available avian genomes has revealed that the foxp3 locus is missing, incomplete or of poor quality in the relevant genomic assemblies for nearly all avian species. Nevertheless, in two species, the peregrine falcon (Falco peregrinus) and the saker falcon (F. cherrug), there is compelling evidence for the existence of exons showing synteny with foxp3 in the ground tit. A broader phylogenomic analysis has shown that FoxP3 sequences from these three species are similar to crocodilian sequences, the closest living relatives of birds. In both birds and crocodilians, we have also identified a highly proline-enriched region at the N terminus of FoxP3, a region previously identified only in mammals
Increased sequencing depth does not increase captured diversity of arbuscular mycorrhizal fungi
The arrival of 454 sequencing represented a major breakthrough by allowing deeper sequencing of environmental samples than was possible with existing Sanger approaches. Illumina MiSeq provides a further increase in sequencing depth but shorter read length compared with 454 sequencing. We explored whether Illumina sequencing improves estimates of arbuscular mycorrhizal (AM) fungal richness in plant root samples, compared with 454 sequencing. We identified AM fungi in root samples by sequencing amplicons of the SSU rRNA gene with 454 and Illumina MiSeq paired-end sequencing. In addition, we sequenced metagenomic DNA without prior PCR amplification. Amplicon-based Illumina sequencing yielded two orders of magnitude higher sequencing depth per sample than 454 sequencing. Initial analysis with minimal quality control recorded five times higher AM fungal richness per sample with Illumina sequencing. Additional quality control of Illumina samples, including restriction of the marker region to the most variable amplicon fragment, revealed AM fungal richness values close to those produced by 454 sequencing. Furthermore, AM fungal richness estimates were not correlated with sequencing depth between 300 and 30,000 reads per sample, suggesting that the lower end of this range is sufficient for adequate description of AM fungal communities. By contrast, metagenomic Illumina sequencing yielded very few AM fungal reads and taxa and was dominated by plant DNA, suggesting that AM fungal DNA is present at prohibitively low abundance in colonised root samples. In conclusion, Illumina MiSeq sequencing yielded higher sequencing depth, but similar richness of AM fungi in root samples, compared with 454 sequencing