speCiaL reviews in ornitHoLogy
Discovery and screening of SNPs generally requires that one obtain sufficient coverage (at least – times), ideally from multiple individuals (often via resequencing), to identify poly- morphisms. is can be a problem for taxa with large genomes (i.e., most eukaryotes) because of the cost required to obtain full genomic sequences at that level of coverage. us, discovery of SNPs can be assisted by reducing the pool of homologous DNA sequences, thereby increasing the coverage level. For instance, the pool can be limited by using only transcribed sequences (the transcriptome or expressed sequence tags [ESTs]), or by restric- tion enzyme digestion of multiple individuals and size selection (Van Tassell et al. ). Vera et al. () used the EST method by pooling cDNA from individuals from eight families of fritillary butterflies. is method produced >, EST se- quences of ∼ bp in average length on the GS-FLX plat- form. ey generated contigs totaling ∼, bp with at least × coverage from the ESTs and were able to identify SNPs, of which involved nonsynonymous polymorphisms. Another recent study (Novaes et al. ) generated Mbp of EST sequence from Eucalyptus grandis cDNA using GS runs and was able to identify , high-confidence SNPs from
, contigs averaging ∼ bp in length. Single-nucleotide
polymorphism microarrays are available for chicken (Burt and White , Schmidt et al. ) and Zebra Finch (Naurin et al. ). With > million SNPs identified from genomic se- quences, microarray analyses with a -kbp SNP array from G. gallus or T. guttata can provide good coverage of the genome for association studies of diverse avian species. Given the in- creased length per read on the with current (and near fu- ture) technology, even greater recovery of SNP sequences is expected, and we believe that application of these NGSMs can greatly enhance studies in population and conservation genetics of birds.
disease diagnosis and analysis
Avian disease is of broad interest because of its potential impact on human health, its importance for conservation of rare spe- cies, and its potential for insight into the coevolution of hosts and pathogens. Because research on infectious diseases is typi- cally well funded and involves relatively small genomes, NGSMs have been applied extensively to sequencing genomes of a range of pathogens. ese include viruses such as avian influenza (Höper et al. ) or avian bornavirus (Gancz et al. ), bacteria such as MRSA Staphylococcus (Highlander et al. ) and avian My- cobacterium (Paustian et al. ), and protozoans such as oxo- plasma gondii (Bontell et al. ). ese methods have also been used in genome-sequencing or genetic-association studies to iden- tify mutations responsible for genetic disorders or diseases (ten Bosch and Grody , Vasta et al. ). In addition, research- ers are beginning to apply NGSMs to diagnosis or identification of pathogenic organisms by random screening (Nakamura et al.
, ; Adams et al. ; Jones et al. ) and by targeted
methods using PCR amplicons (Jordan et al. ). Targeted, tagged pyrosequencing methods have also been used to assess variation in host immune-system genes, such as those of the MHC (Babik et al. , Bentley et al. , Wiseman et al. ) and immunoglobulins (Glanville et al. ). us far, very few NGSM studies have addressed pathogen–host relationships within birds,
with the exception of a few studies on avian influenza (e.g., Höper et al. ) or diseases of domestic fowl (e.g., Paustian et al. , Spatz and Rue , Gancz et al. ).
Gene expression and transcriptome analyses
One of the most powerful new applications of NGSMs is in mea- suring gene expression (Nielsen et al. , Torres et al. , Mo- rozova et al. ). Most NGSMs produce high average coverage (i.e., number of sequence copies) per nucleotide site, providing an estimation of the frequency of any particular DNA molecule in an overall DNA pool. In most expression analyses, mRNA is isolated from developing organs or from tissues that have undergone dif- ferential treatment (e.g., pathogen-infected vs. non-infected indi- viduals), and the RNA is reverse transcribed in vitro into cDNA. With appropriate experimental controls and an assumption that the reverse-transcription process does not alter the relative fre- quencies of the various mRNA transcripts, the relative depth of coverage of the sequence should be proportional to the expres- sion of the particular gene. Some early analyses (e.g., Torres et al.
) suggested that there were biases in the representation of
certain, usually smaller, transcript sequences. But later analyses showed ways to avoid (e.g., size standardization by nebulization) or correct for these biases and revealed NGSMs as a powerful tool for gene expression and evo-devo studies (Weber et al. , Mo- rozova et al. ). Direct RNA sequencing (i.e., without reverse transcription to cDNA) is a promising new application of the Heli- cos platform (Table ) that will surely advance gene expression and transcriptome analyses.
Next-generation sequencing methods are well positioned to replace the use of more standard methods for measuring gene ex- pression (Northern blots, real-time RT-PCR, or microarray analy- sis). We anticipate their use in studies of avian development such as those recently conducted by Abzhanov et al. (, ). ese studies revealed, via comparative evaluations of expression levels of candidate genes in developing beak tissues and microarray anal- yses of expression, that the gene Bmp is up-regulated in Darwin’s finches with larger, thicker bills and that the calmodulin gene is up-regulated in Darwin’s finches with longer, narrower bills. We also expect to see their use in studies that evaluate changes in gene expression in hosts or vectors in response to infection by avian pathogens, such as avian malaria or avian influenza, much as has been done for pathogens of agricultural or medical importance (e.g., chestnut blight; Barakat et al. ).
Some of the earliest applications of next-generation sequencing involved sequencing of ancient materials such as subfossil bones, hair from mammals, and ancient soil samples (Poinar et al. , Gilbert et al. ). Initially, these studies involved random, shot- gun sequencing, but soon targeted amplification was applied to obtain full mitochondrial genomes from ancient samples (Briggs et al. , Stiller et al. ). Most of the applications in verte- brates, thus far, have involved mammals (e.g., Noonan et al. ,
; Poinar et al. ; Willerslev et al. ), the only published
use with ancient avian materials being a study on moa microsatel- lite development (see above; Allentoft et al. ).
A recent phylogenetic study (Gilbert et al. ) showed that sequencing of full mtDNA genomes with pyrosequencing