A feature selection approach for identification of signature genes from SAGE data

Background  

One goal of gene expression profiling is to identify signature genes that robustly distinguish different types or grades of tumors. Several tumor classifiers based on expression profiling have been proposed using microarray technique. Due to important differences in the probabilistic models of microarray and SAGE technologies, it is important to develop suitable techniques to select specific genes from SAGE measurements.     

Incongruence between morphological and molecular markers in the butterfly genus Zizina (Lepidoptera: Lycaenidae) in New Zealand

The butterfly genus Zizina in New Zealand has a complex taxonomic history due to the presence of morphological intermediates between the two species, the endemic Z. oxleyi and the introduced Z. labradus, in a putative hybrid zone on the east coast of the South Island of New Zealand. This makes species identification in the field problematic, particularly as the presence of hybrids has not been confirmed. We address this uncertainty through morphological and molecular analyses. Specimens were collected from a range of locations in New Zealand, as well as from Australia, and measurements were made of male genitalia and ventral wing coloration. Two mitochondrial genes (COI, ND5) and three nuclear gene fragments (28S, ITS2 and wingless) were also sequenced for a selection of individuals, and the presence of Wolbachia species in genomic DNA was tested. The two species were separable in morphological space, although there was some overlap, and the contact zone appeared to be around Kaikoura on the east coast of the South Island. Furthermore, specimens from the putative hybrid zone could be classified as Z. oxleyi using morphological characters individually, but not when these were used in a principal component analysis. Molecular analysis showed that there was a mean sequence divergence of 2.0% between two clades for COI, and 4.1% for ND5, but suggested that the contact zone between them was in the north‐west of the South Island. However, there was only a single clade for each of the three nuclear markers. It is thought that this incongruence between morphological and molecular markers is indicative of hybridization which is more extensive than previously thought. However, the possibility that recent speciation has occurred or is occurring is not ruled out.     

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples

Background

Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA.

Results

The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585).The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing.

Conclusion

This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.     

Bayesian adaptive sequence alignment algorithms

The selection of a scoring matrix and gap penalty parameters continues to be an important problem in sequence alignment. We describe here an algorithm, the 'Bayes block aligner, which bypasses this requirement. Instead of requiring a fixed set of parameter settings, this algorithm returns the Bayesian posterior probability for the number of gaps and for the scoring matrices in any series of interest. Furthermore, instead of returning the single best alignment for the chosen parameter settings, this algorithm returns the posterior distribution of all alignments considering the full range of gapping and scoring matrices selected, weighing each in proportion to its probability based on the data. We compared the Bayes aligner with the popular Smith-Waterman algorithm with parameter settings from the literature which had been optimized for the identification of structural neighbors, and found that the Bayes aligner correctly identified more structural neighbors. In a detailed examination of the alignment of a pair of kinase and a pair of GTPase sequences, we illustrate the algorithm's potential to identify subsequences that are conserved to different degrees. In addition, this example shows that the Bayes aligner returns an alignment-free assessment of the distance between a pair of sequences.      

The effects of trout on epibenthic odonate naiads in stream pools

• 1 In a southern Califomian stream naiads of a lestid damselfly (Archilestes grandis Rambur) were much less abundant, moved less, exhibited fewer conspicuous behaviours, were more likely to occur in refuge areas, and had different diets in pools containing versus pools lacking rainbow trout (Oncorhynchus mykiss Richardson). To determine if trout were responsible for these patterns, we removed trout from some stream pools and added them to pools lacking trout, with unmanipulated trout and troutless pools acting as controls.
• 2 The abundance and emergence of A. grandis were drastically reduced, and the proportion of lestid populations in refuge areas greatly increased, when trout were added to pools; however, the removal of trout had less drastic effects on lestid abundance and distribution, Aeshna walkeri (Kennedy) was also more abundant in pools lacking than in pools containing trout.
• 3 Trout manipulations affected lestid behaviour, with swimming being observed only in troutless pools and movement tending to be greater in pools lacking rather than containing trout.
• 4 One week after manipulations started, the number of prey items per lestid gut was higher in troutless control than in trout addition pools. Ostracods and chironomids were more abundant, and mayflies were less abundant, in the diets of lestids from pools lacking versus containing trout. Comparisons of the environmental abundances of prey taxa and lestid diet composition indicated that lestid selectivities for Caenis were higher, and those for Paraleptophlebia, ostracods, and Eubrianax lower, in trout than in troutless pools.
• 5 Although similar at the beginning of manipulations, head widths of lestids in troutless control pools were greater than those in trout addition pools after 3 weeks.

     

Evidence from nuclear sequences that invariable sites should be considered when sequence divergence is calculated

It has long been known, from the distribution of multiple amino acid replacements, that not all amino acids of a sequence are replaceable. More recently, the phenomenon was observed at the nucleotide level in mitochondrial DNA even after allowing for different rates of transition and transversion substitutions. We have extended the search to globin gene sequences from various organisms, with the following results: (1) Nearly every data set showed evidence of invariable nucleotide positions. (2) In all data sets, substitution rates of transversions and transitions were never in the ratio of 2/1, and rarely was the ratio even constant. (3) Only rarely (e.g., the third codon position of beta hemoglobins) was it possible to fit the data set solely by making allowance for the number of invariable positions and for the relative rates of transversion and transition substitutions. (4) For one data set (the second codon position of beta hemoglobins) we were able to simulate the observed data by making the allowance in (3) and having the set of covariotides (concomitantly variable nucleotides) be small in number and be turned over in a stochastic manner with a probability that was appreciable. (5) The fit in the latter case suggests, if the assumptions are correct and at all common, that current procedures for estimating the total number of nucleotide substitutions in two genes since their divergence from their common ancestor could be low by as much as an order of magnitude. (6) The fact that only a small fraction of the nucleotide positions differ is no guarantee that one is not seriously underestimating the total amount of divergence (substitutions). (7) Most data sets are so heterogeneous in their number of transition and transversion differences that none of the current models of nucleotide substitution seem to fit them even after (a) segregation of coding from noncoding sequences and (b) splitting of the codon into three subsets by codon position. (8) These frequently occurring problems cannot be seen unless several reasonably divergent orthologous genes are examined together.      

Effects of plant height and row spacing on kenaf forage potential with multiple harvests

Kenaf (Hibiscus cannabinus L.) forage potential can be enhanced through its regrowth capacity and higher production in narrow rows. A field experiment was conducted in Matamoros, Coahuila, Mexico, during 2 growing seasons (2004 and 2005) to study the effects of plant height and row spacing on kenaf forage potential with multiple harvests. This study evaluated the effects of (1) 2 plant heights at cutting (1.0-1.2 m and 1.8-2.0 m) and (2) 4 inter row spacings (0.19, 0.38, 0.57 and 0.76 m) using a 2 x 4 factorial arrangement of treatments in a completely randomized block design with 4 replications. Dry matter (DM) and crude protein (CP) yields, DM partitioning, neutral detergent fiber (NDF) and CP concentrations were determined. Heights at cutting × row spacing interactions were not significant for the monitored variables (p>0.05). Kenaf response to treatments was only relevant for main effects (p≤0.05). Row spacing and plant height affected DM and CP yields (p≤0.05), whereas only plant height affected chemical composition and DM partitioning (p≤0.05). Dry matter (17.0%-26.0%), and CP (12.4%-15.6%) yields were higher (p≤0.05) when plant heights had reached 1.8 to 2.0 m. Row spacing reduction from 0.76 m to 0.38 and 0.19 m increased DM yield (20.4-33.4%) and CP yield (24.2-38.5%) (p≤0.05). Kenaf forage potential increases when planted in narrow rows and harvested 2 or 3 times during the growing season.