Development and diversity of Andean-derived, gene-based microsatellites for common bean (Phaseolus vulgaris L.)

Background  

Gene-based (genic) microsatellites are a useful tool for plant genetics and simple sequence repeat loci can often be found in coding regions of the genome. While EST sequencing can be used to discover genic microsatellites, direct screening of cDNA libraries for repeat motifs can save on overall sequencing costs. The objective of this research was to screen a large cDNA library from and Andean common bean genotype for six di-nucleotide and tri-nucleotide repeat motifs through a filter hybridization approach and to develop microsatellite markers from positive clones.     

SAT,a flexible and optimized Web application for SSR marker development

Background  

Simple Sequence Repeats (SSRs), or microsatellites, are among the most powerful genetic markers known. A common method for the development of SSR markers is the construction of genomic DNA libraries enriched for SSR sequences, followed by DNA sequencing. However, designing optimal SSR markers from bulk sequence data is a laborious and time-consuming process.     

Rapid evolution of mammalian X-linked testis microRNAs

Background

Microsatellites in cDNA are useful as molecular markers because they represent transcribed genes and can be used as anchor markers for linkage and comparative mapping, as well as for studying genome evolution. Microsatellites in cDNA can be detected in existing ESTs by data mining. However, in most fish species, no ESTs are available or the number of ESTs is limited, although fishes represent half of the vertebrates on the earth. We developed a simple and efficient method for isolation of microsatellites from cDNA in fish.

Results

The method included normalization of 150 ng cDNA using 0.5 U duplex-specific nuclease (DSN) at 65°C for 30 min, enrichment of microsatellites using biotinylated oligonucleotides and magnetic field, and directional cloning of cDNA into a vector. We tested this method to enrich CA- and GA-microsatellites from cDNA of Asian seabass, and demonstrated that enrichment of microsatellites from normalized cDNA could increased the efficiency of microsatellite isolation over 30 times as compared to direct sequencing of clones from cDNA libraries. One hundred and thirty-nine (36.2%) out of 384 clones from normalized cDNA contained microsatellites. Unique microsatellite sequences accounted for 23.6% (91/384) of sequenced clones. Sixty microsatellites isolated from cDNA were characterized, and 41 were polymorphic. The average allele number of the 41 microsatellites was 4.85 ± 0.54, while the expected heterozygosity was 0.56 ± 0.03. All the isolated microsatellites inherited in a Mendelian pattern.

Conclusion

Normalization of cDNA substantially increased the efficiency of enrichment of microsatellites from cDNA. The described method for isolation of microsatellites from cDNA has the potential to be applied to a wide range of fish species. The microsatellites isolated from cDNA could be useful for linkage and comparative mapping, as well as for studying genome evolution.     

Negative Subtraction Hybridization: An efficient method to isolate large numbers of condition-specific cDNAs

Background  

The construction of cDNA libraries is a useful tool to understand gene expression in organisms under different conditions, but random sequencing of unbiased cDNA collections is laborious and can give rise to redundant EST collections.     

Errors in CGAP xProfiler and cDNA DGED: the importance of library parsing and gene selection algorithms

Background  

The Cancer Genome Anatomy Project (CGAP) xProfiler and cDNA Digital Gene Expression Displayer (DGED) have been made available to the scientific community over a decade ago and since then were used widely to find genes which are differentially expressed between cancer and normal tissues. The tissue types are usually chosen according to the ontology hierarchy developed by NCBI. The xProfiler uses an internally available flat file database to determine the presence or absence of genes in the chosen libraries, while cDNA DGED uses the publicly available UniGene Expression and Gene relational databases to count the sequences found for each gene in the presented libraries.     

An EST resource for tilapia based on 17 normalized libraries and assembly of 116,899 sequence tags

Background

Large collections of expressed sequence tags (ESTs) are a fundamental resource for analysis of gene expression and annotation of genome sequences. We generated 116,899 ESTs from 17 normalized and two non-normalized cDNA libraries representing 16 tissues from tilapia, a cichlid fish widely used in aquaculture and biological research.

Results

The ESTs were assembled into 20,190 contigs and 36,028 singletons for a total of 56,218 unique sequences and a total assembled length of 35,168,415 bp. Over the whole project, a unique sequence was discovered for every 2.079 sequence reads. 17,722 (31.5%) of these unique sequences had significant BLAST hits (e-value < 10^-10) to the UniProt database.

Conclusion

Normalization of the cDNA pools with double-stranded nuclease allowed us to efficiently sequence a large collection of ESTs. These sequences are an important resource for studies of gene expression, comparative mapping and annotation of the forthcoming tilapia genome sequence.

     

ProLoc-GO: Utilizing informative Gene Ontology terms for sequence-based prediction of protein subcellular localization

Background  

Gene Ontology (GO) annotation, which describes the function of genes and gene products across species, has recently been used to predict protein subcellular and subnuclear localization. Existing GO-based prediction methods for protein subcellular localization use the known accession numbers of query proteins to obtain their annotated GO terms. An accurate prediction method for predicting subcellular localization of novel proteins without known accession numbers, using only the input sequence, is worth developing.     

SMART amplification combined with cDNA size fractionation in order to obtain large full-length clones

Background  

cDNA libraries are widely used to identify genes and splice variants, and as a physical resource for full-length clones. Conventionally-generated cDNA libraries contain a high percentage of 5'-truncated clones. Current library construction methods that enrich for full-length mRNA are laborious, and involve several enzymatic steps performed on mRNA, which renders them sensitive to RNA degradation. The SMART technique for full-length enrichment is robust but results in limited cDNA insert size of the library.     

Heart-specific genes revealed by expressed sequence tag (EST) sampling

Background  

Cardiovascular diseases are the primary cause of death worldwide; the identification of genes specifically expressed in the heart is thus of major biomedical interest. We carried out a comprehensive analysis of gene-expression profiles using expressed sequence tags (ESTs) to identify genes overexpressed in the human adult heart. The initial set of genes expressed in the heart was constructed by clustering and assembling ESTs from heart cDNA libraries. Expression profiles were then generated for each gene by counting their cognate ESTs in all libraries. Differential expression was assessed by applying a previously published statistical procedure to these profiles.     

Poly: a quantitative analysis tool for simple sequence repeat (SSR) tracts in DNA

Background  

Simple sequence repeats (SSRs), microsatellites or polymeric sequences are common in DNA and are important biologically. From mononucleotide to trinucleotide repeats and beyond, they can be found in long (> 6 repeating units) tracts and may be characterized by quantifying the frequencies in which they are found and their tract lengths. However, most of the existing computer programs that find SSR tracts do not include these methods.