Low conservation of alternative splicing patterns in the human and mouse genomes

Alternative splicing has recently emerged as a major mechanism of generating protein diversity in higher eukaryotes. We compared alternative splicing isoforms of 166 pairs of orthologous human and mouse genes. As the mRNA and EST libraries of human and mouse are not complete and thus cannot be compared directly, we instead analyzed whether known cassette exons or alternative splicing sites from one genome are conserved in the other genome. We demonstrate that about half of the analyzed genes have species-specific isoforms, and about a quarter of elementary alternatives are not conserved between the human and mouse genomes. The detailed results of this study are available at www.ig-msk.ru:8005/HMG_paper.     

Identification of evolutionary hotspots in the rodent genomes

We describe a whole-genome comparative analysis of the human, mouse, and rat genomes to describe the average substitution patterns of four genomic regions: ancient repeats, rodent-specific DNA, exons, and conserved (coding and noncoding) regions, and to identify rodent evolutionary hotspots. In all types of regions, except the rodent-specific DNA, the rat branch is slightly longer than the mouse branch. Moreover, the mouse-rat distance is longer in the rodent-specific DNA than in the ancient repeats. Analysis of individual conserved regions with different substitution models yielded the conclusion that the Jukes-Cantor model is inadequate, and the Hasegawa-Kishino-Yano model is almost as good as the REV model. Using human as an outgroup, we identified 5055 evolutionary hotspots, which are highly conserved subalignment blocks (each consisting of at least 100 aligned sites and a small fraction of gaps) with a large and statistically significant difference in the branch lengths of the rodent species. The cutoffs used to identify the hotspots are partially based on estimates of the average rates of substitution. The fractions of hotspots overlapping with the rodent RefSeq genes, RefSeq exons, and ESTs are all higher than expected. Still, more than half of the hotspots lie in noncoding regions of the mouse genome. We believe that the hotspots represent biologically interesting regions in the rodent genomes.     

Genome-Scale Evolution: Reconstructing Gene Orders in the Ancestral Species

Recent progress in genome-scale sequencing and comparative mapping raises new challenges in studies of genome rearrangements. Although the pairwise genome rearrangement problem is well-studied, algorithms for reconstructing rearrangement scenarios for multiple species are in great need. The previous approaches to multiple genome rearrangement problem were largely based on the breakpoint distance rather than on a more biologically accurate rearrangement (reversal) distance. Another shortcoming of the existing software tools is their inability to analyze rearrangements (inversions, translocations, fusions, and fissions) of multichromosomal genomes. This paper proposes a new multiple genome rearrangement algorithm that is based on the rearrangement (rather than breakpoint) distance and that is applicable to both unichromosomal and multichromosomal genomes. We further apply this algorithm for genome-scale phylogenetic tree reconstruction and deriving ancestral gene orders. In particular, our analysis suggests a new improved rearrangement scenario for a very difficult Campanulaceae cpDNA dataset and a putative rearrangement scenario for human, mouse and cat genomes.     

PCBP2基因座位上长非编码RNA的筛选与鉴定

目的从EST鉴定入手利用生物信息学方法筛选验证PCBP2基因内部UC.338基因座位附近的长非编码RNA。方法首先利用生物信息学方法筛选包含内含子转录区域的ESTs,经CPC分析和统计学比较,共筛选到4个(人、鼠各两个)可能的lncRNAs,用PCR方法进行鉴定,克隆到pGEM-T载体测序验证,并用PCR,real-time PCR的方法检测各ESTs在人源细胞系或小鼠不同组织,小鼠发育不同时间点大脑中的表达谱。结果筛选到4个ESTs中有3个ESTs均具有一定的非编码特征,并最终测序正确:1个人源,2个鼠源,并且其中有些lncRNAs具有一定的细胞、组织特异性,有些lncRNAs具有广谱表达模式。结论使用了一种新的寻找lncRNAs的方法,从EST鉴定入手,对现有数据库进行了挖掘。找到了可能在小鼠大脑发育过程中起作用的lncRNAs。     

Selecting for functional alternative splices in ESTs

The expressed sequence tag (EST) collection in dbEST provides an extensive resource for detecting alternative splicing on a genomic scale. Using genomically aligned ESTs, a computational tool (TAP) was used to identify alternative splice patterns for 6400 known human genes from the RefSeq database. With sufficient EST coverage, one or more alternatively spliced forms could be detected for nearly all genes examined. To identify high (>95%) confidence observations of alternative splicing, splice variants were clustered on the basis of having mutually exclusive structures, and sample statistics were then applied. Through this selection, alternative splices expected at a frequency of >5% within their respective clusters were seen for only 17%-28% of genes. Although intron retention events (potentially unspliced messages) had been seen for 36% of the genes overall, the same statistical selection yielded reliable cases of intron retention for <5% of genes. For high-confidence alternative splices in the human ESTs, we also noted significantly higher rates both of cross-species conservation in mouse ESTs and of validation in the GenBank mRNA collection. We suggest quantitative analytical approaches such as these can aid in selecting useful targets for further experimental characterization and in so doing may help elucidate the mechanisms and biological implications of alternative splicing.     

Efficient de novo assembly of large genomes using compressed data structures

De novo genome sequence assembly is important both to generate new sequence assemblies for previously uncharacterized genomes and to identify the genome sequence of individuals in a reference-unbiased way. We present memory efficient data structures and algorithms for assembly using the FM-index derived from the compressed Burrows-Wheeler transform, and a new assembler based on these called SGA (String Graph Assembler). We describe algorithms to error-correct, assemble, and scaffold large sets of sequence data. SGA uses the overlap-based string graph model of assembly, unlike most de novo assemblers that rely on de Bruijn graphs, and is simply parallelizable. We demonstrate the error correction and assembly performance of SGA on 1.2 billion sequence reads from a human genome, which we are able to assemble using 54 GB of memory. The resulting contigs are highly accurate and contiguous, while covering 95% of the reference genome (excluding contigs <200 bp in length). Because of the low memory requirements and parallelization without requiring inter-process communication, SGA provides the first practical assembler to our knowledge for a mammalian-sized genome on a low-end computing cluster.     

Intronic sequences flanking alternatively spliced exons are conserved between human and mouse

Comparison of the sequences of mouse and human genomes revealed a surprising number of nonexonic, nonexpressed conserved sequences, for which no function could be assigned. To study the possible correlation between these conserved intronic sequences and alternative splicing regulation, we developed a method to identify exons that are alternatively spliced in both human and mouse. We compiled two exon sets: one of alternatively spliced conserved exons and another of constitutively spliced conserved exons. We found that 77% of the conserved alternatively spliced exons were flanked on both sides by long conserved intronic sequences. In comparison, only 17% of the conserved constitutively spliced exons were flanked by such conserved intronic sequences. The average length of the conserved intronic sequences was 103 bases in the upstream intron and 94 bases in the downstream intron. The average identity levels in the immediately flanking intronic sequences were 88% and 80% for the upstream and downstream introns, respectively, higher than the conservation levels of 77% that were measured in promoter regions. Our results suggest that the function of many of the intronic sequence blocks that are conserved between human and mouse is the regulation of alternative splicing.     

Gene inactivation and its implications for annotation in the era of personal genomics

The first wave of personal genomes documents how no single individual genome contains the full complement of functional genes. Here, we describe the extent of variation in gene and pseudogene numbers between individuals arising from inactivation events such as premature termination or aberrant splicing due to single-nucleotide polymorphisms. This highlights the inadequacy of the current reference sequence and gene set. We present a proposal to define a reference gene set that will remain stable as more individuals are sequenced. In particular, we recommend that the ancestral allele be used to define the reference sequence from which a core human reference gene annotation set can be derived. In addition, we call for the development of an expanded gene set to include human-specific genes that have arisen recently and are absent from the ancestral set.     

Comparative gene prediction in human and mouse

The completion of the sequencing of the mouse genome promises to help predict human genes with greater accuracy. While current ab initio gene prediction programs are remarkably sensitive (i.e., they predict at least a fragment of most genes), their specificity is often low, predicting a large number of false-positive genes in the human genome. Sequence conservation at the protein level with the mouse genome can help eliminate some of those false positives. Here we describe SGP2, a gene prediction program that combines ab initio gene prediction with TBLASTX searches between two genome sequences to provide both sensitive and specific gene predictions. The accuracy of SGP2 when used to predict genes by comparing the human and mouse genomes is assessed on a number of data sets, including single-gene data sets, the highly curated human chromosome 22 predictions, and entire genome predictions from ENSEMBL. Results indicate that SGP2 outperforms purely ab initio gene prediction methods. Results also indicate that SGP2 works about as well with 3x shotgun data as it does with fully assembled genomes. SGP2 provides a high enough specificity that its predictions can be experimentally verified at a reasonable cost. SGP2 was used to generate a complete set of gene predictions on both the human and mouse by comparing the genomes of these two species. Our results suggest that another few thousand human and mouse genes currently not in ENSEMBL are worth verifying experimentally.     

The Ensembl automatic gene annotation system

As more genomes are sequenced, there is an increasing need for automated first-pass annotation which allows timely access to important genomic information. The Ensembl gene-building system enables fast automated annotation of eukaryotic genomes. It annotates genes based on evidence derived from known protein, cDNA, and EST sequences. The gene-building system rests on top of the core Ensembl (MySQL) database schema and Perl Application Programming Interface (API), and the data generated are accessible through the Ensembl genome browser (http://www.ensembl.org). To date, the Ensembl predicted gene sets are available for the A. gambiae, C. briggsae, zebrafish, mouse, rat, and human genomes and have been heavily relied upon in the publication of the human, mouse, rat, and A. gambiae genome sequence analysis. Here we describe in detail the gene-building system and the algorithms involved. All code and data are freely available from http://www.ensembl.org.