Low-quality Sequences Research Articles

Sequence-indexed mutations in maize using the UniformMu transposon-tagging population

BackgroundGene knockouts are a critical resource for functional genomics. In Arabidopsis, comprehensive knockout collections were generated by amplifying and sequencing genomic DNA flanking insertion mutants. These Flanking Sequence Tags (FSTs) map each mutant to a specific locus within the genome. In maize, FSTs have been generated using DNA transposons. Transposable elements can generate unstable insertions that are difficult to analyze for simple knockout phenotypes. Transposons can also generate somatic insertions that fail to segregate in subsequent generations.ResultsTransposon insertion sites from 106 UniformMu FSTs were tested for inheritance by locus-specific PCR. We confirmed 89% of the FSTs to be germinal transposon insertions. We found no evidence for somatic insertions within the 11% of insertion sites that were not confirmed. Instead, this subset of insertion sites had errors in locus-specific primer design due to incomplete or low-quality genomic sequences. The locus-specific PCR assays identified a knockout of a 6-phosphogluconate dehydrogenase gene that co-segregates with a seed mutant phenotype. The mutant phenotype linked to this knockout generates novel hypotheses about the role for the plastid-localized oxidative pentose phosphate pathway during grain-fill.ConclusionWe show that FSTs from the UniformMu population identify stable, germinal insertion sites in maize. Moreover, we show that these sequence-indexed mutations can be readily used for reverse genetic analysis. We conclude from these data that the current collection of 1,882 non-redundant insertion sites from UniformMu provide a genome-wide resource for reverse genetics.

An SNP resource for rice genetics and breeding based on subspecies indica and japonica genome alignments.

Dense coverage of the rice genome with polymorphic DNA markers is an invaluable tool for DNA marker-assisted breeding, positional cloning, and a wide range of evolutionary studies. We have aligned drafts of two rice subspecies, indica and japonica, and analyzed levels and patterns of genetic diversity. After filtering multiple copy and low quality sequence, 408,898 candidate DNA polymorphisms (SNPs/INDELs) were discerned between the two subspecies. These filters have the consequence that our data set includes only a subset of the available SNPs (in particular excluding large numbers of SNPs that may occur between repetitive DNA alleles) but increase the likelihood that this subset is useful: Direct sequencing suggests that 79.8% +/- 7.5% of the in silico SNPs are real. The SNP sample in our database is not randomly distributed across the genome. In fact, 566 rice genomic regions had unusually high (328 contigs/48.6 Mb/13.6% of genome) or low (237 contigs/64.7 Mb/18.1% of genome) polymorphism rates. Many SNP-poor regions were substantially longer than most SNP-rich regions, covering up to 4 Mb, and possibly reflecting introgression between the respective gene pools that may have occurred hundreds of years ago. Although 46.2% +/- 8.3% of the SNPs differentiate other pairs of japonica and indica genotypes, SNP rates in rice were not predictive of evolutionary rates for corresponding genes in another grass species, sorghum. The data set is freely available at http://www.plantgenome.uga.edu/snp.

ESTAnnotator: A tool for high throughput EST annotation.

In high throughput sequence analysis, it is often necessary to combine the results of contemporary bioinformatics tools, because no individual tool alone computes all the requested information. ESTAnnotator is a tool for the high throughput annotation of expressed sequence tags (ESTs) by automatically running a collection of bioinformatics applications. In the first step, a quality check is performed and repeats, vector parts and low quality sequences are masked. Then successive steps of database searching and EST clustering are performed. Already known transcripts present within mRNA and genomic DNA reference databases are identified. Subsequently, tools for the clustering of anonymous ESTs, and for further database searches at the protein level, are applied. Finally, the outputs of each individual tool are gathered and the relevant results presented in a descriptive summary. ESTAnnotator was already successfully applied for the systematic identification and characterisation of novel human genes involved in cartilage/bone formation, growth, differentiation and homeostasis. ESTAnnotator is available at http://genome.dkfz-heidelberg.de, contact: genome@dkfz.de.

Human Full-Length Pre-mRNA Sequence Dataset for Computational Gene Prediction and Alternative Splicing Analysis

Computational gene prediction has recently become essential to identify all the genes from enormousgenome sequencesandto de netheir functions. However, gene prediction methodsstill showlow speci- city (45% at the exon level) [7]. Although computationally predicted data by automatic annotationsystem are growing rapidly, experimentally veri ed data provide the cornerstone for improvements ofgene prediction. Because there is no guarantee that the predicted events occur in vivo. It has becomeimportant to discriminate experimentally veri ed data from computationally predicted data [5].Aligning expressed sequence tags (ESTs)/mRNAs to the genomic sequences has been a practicalapproach to detect gene regions and to identify alternative splicing on a genomic scale. In previousstudies EST-genome alignments were made using 90-93% sequence identity as threshold [2, 3, 4].However, their low thresholds allow ESTs to incorrectly align with paralogous genes or pseudogenes.Moreover, EST sequences have low-quality sequencing region or contaminated region [6].We propose to collect true data in the view of quality rather than quantity for furtherimprovementof gene prediction and functional analysis. In this study, we retrieved human full-length pre-mRNA(FL-pre-mRNA) sequences by parsing experimentally veri ed annotation in GenBank records, andthen con rmed complete length and exon-intron boundaries (splice sites) in the sequences by multipleEST alignment. Additionally, we identi ed and classi ed constitutive and alternative splice sites onthe sequences. Moreover, we demonstrate novel ndings about human gene structure and splice sitesbased on this high-quality dataset.

Escherichia coli single-stranded DNA-binding protein, a molecular tool for improved sequence quality in pyrosequencing.

Pyrosequencing is a four-enzyme bioluminometric DNA sequencing technique based on a DNA sequencing by synthesis principle. Currently, the technique is limited to analysis of short DNA sequences exemplified by single-nucleotide polymorphism analysis. In order to expand the field for pyrosequencing, the read length needs to be improved and efforts have been made to purify reaction components as well as add single-stranded DNA-binding protein (SSB) to the pyrosequencing reaction. In this study, we have performed a systematic effort to analyze the effects of SSB by comparing the pyrosequencing result of 103 independent complementary DNA (cDNA) clones. More detailed information about the cause of low quality sequences on templates with different characteristics was achieved by thorough analysis of the pyrograms. Also, real-time biosensor analysis was performed on individual cDNA clones for investigation of primer annealing and SSB binding on these templates. Results from these studies indicate that templates with high performance in pyrosequencing without SSB possess efficient primer annealing and low SSB affinity. Alternative strategies to improve the performance in pyrosequencing by increasing the primer-annealing efficiency have also been evaluated.

Finding Homologs to Nucleic Acid or Protein Sequences Using the Framesearch Program

The Framesearch algorithm includes the possibility of a frameshift error in its alignment algorithm, and therefore can find alignments that span different reading frames. Protocols in this unit describe the use of Framesearch to search a protein sequence database for sequences that are similar to a query nucleotide sequence, and to search a nucleotide sequence database for sequences that are similar to a query protein sequence. Three alternate protocols describe ways to improve the speed of Framesearch and thus make it practical for routine use. Framesearch is especially appropriate for low-quality single-read nucleotide sequence data, such as ESTs (expressed sequence tags) or early drafts of genomic sequences; it does not offer any significant advantage over less CPU-intensive algorithms for relatively high-quality nucleotide sequences without many single-nucleotide insertion or deletion errors.

Transcriptome analysis of channel catfish ( Ictalurus punctatus): initial analysis of gene expression and microsatellite-containing cDNAs in the skin

Previous molecular genetic studies on channel catfish ( Ictalurus punctatus) have focused on limited number of genes and gene products. Recent advancement of molecular techniques made high throughput analysis of transcriptomes possible. As part of our transcriptome analysis of channel catfish, we have analyzed 1909 expressed sequence tags (ESTs) derived from a skin library. Of the 1909 ESTs analyzed, 1376 (72.1%) ESTs representing 496 unique genes had homologies with other organisms while 478 (25.0%) ESTs had no significant homologies and were designated as unknown. The remaining 55 (2.9%) EST clones were eliminated because of their low quality or short sequences. Of the 496 unique genes, 327 (65.9%) genes were singletons while 169 (34.1%) genes represented by two or more ESTs. A total of 1007 (52.8%) ESTs representing 235 unique genes matched previously reported channel catfish ESTs while 847 (44.4%) ESTs representing 261 unique genes were newly identified from this research. Functional categorization of the channel catfish genes indicated that the largest group was ribosomal proteins with 65 unique genes represented by 500 clones. The most abundantly expressed gene, the calcium binding protein ictacalcin, accounted for almost 5% of overall expression, indicating its important function in the skin. Sequence analysis of ESTs revealed the presence of 89 microsatellite-containing genes that may be valuable for future mapping studies.

The libraries that made SUCEST

A large-scale sequencing of sugarcane expressed sequence tags (ESTs) was carried out as a first step in depicting the genome of this important tropical crop. Twenty-six unidirectional cDNA libraries were constructed from a variety of tissues sampled from thirteen different sugarcane cultivars. A total of 291,689 cDNA clones were sequenced in their 5’ and 3’end regions. After trimming low-quality sequences and removing vector and ribosomal RNA sequences, 237,954 ESTs potentially derived from protein-encoding messenger RNA (mRNA) remained. The average insert size in all libraries was estimated to be 1,250bp with the insert length varying from 500 to 5,000 bp. Clustering the 237,954 sugarcane ESTs resulted in 43,141clusters, from which 38% had no matches with existing sequences in the public databases. Around 53% of the clusters were formed by ESTs expressed in at least two libraries while 47% of the clusters are formed by ESTs expressed in only one library. A global analysis of the ESTs indicated that around 33% contain cDNA clones with full-length insert.

Microbial genomics

Alexei Sorokin and colleagues at the Institut National de la Recherche Agronomique, Jouy en Josas, France, have just reported a low-quality (∼1% error) contiguous sequence of the entire 2.36-Mb genome of Lactococcus lactis IL1403 (http://spock. jouy.inra.fr.) 1 Bolotin A. et al. Low-redundancy sequencing of the entire Lactococcus lactis IL1403 genome. Antonie Van Leeuwenhoek. 1999; 76: 27-76 Crossref PubMed Scopus (158) Google Scholar . L. lactis is an AT-rich Gram-positive microorganism used in the cheese industry, which is also used as a model organism for studying the physiology of lactic acid bacteria. Interestingly, Sorokin et al. supplemented the currently fashionable random shotgun sequencing approach with a range of techniques including multiplex long accurate PCR mapping and primer walking on a variety of templates. Analysis of the genome sequence confirmed the presence of at least four prophages in the chromosome of IL1403, identified ∼40 copies of insertion elements, 15 of which represent an IS1070-like element previously unknown in L. lactis, and identified homologues of all the late competence genes in Bacillus subtilis.