Genome-wide analysis of single-nucleotide polymorphisms in human expressed sequences

Single-nucleotide polymorphisms (SNPs) have been explored as a high-resolution marker set for accelerating the mapping of disease genes. Here we report 48,196 candidate SNPs detected by statistical analysis of human expressed sequence tags (ESTs), associated primarily with coding regions of genes. We used Bayesian inference to weigh evidence for true polymorphism versus sequencing error, misalignment or ambiguity, misclustering or chimaeric EST sequences, assessing data such as raw chromatogram height, sharpness, overlap and spacing, sequencing error rates, context-sensitivity and cDNA library origin. Three separate validations-comparison with 54 genes screened for SNPs independently, verification of HLA-A polymorphisms and restriction fragment length polymorphism (RFLP) testing-verified 70%, 89% and 71% of our predicted SNPs, respectively. Our method detects tenfold more true HLA-A SNPs than previous analyses of the EST data. We found SNPs in a large fraction of known disease genes, including some disease-causing mutations (for example, the HbS sickle-cell mutation). Our comprehensive analysis of human coding region polymorphism provides a public resource for mapping of disease genes (available at http://www.bioinformatics.ucla.edu/snp).     

Single pass sequencing and physical and genetic mapping of human brain cDNAs.

We have performed single pass sequencing of 1,024 human brain cDNAs, over 900 of which seem to represent new human genes. Library prescreening with total brain cDNA significantly reduced repeated sequencing of highly represented cDNAs. A subset of sequenced cDNAs were physically mapped to their chromosomal locations using gene-specific STS primers derived from 3' untranslated regions. We have also determined that human brain cDNAs represent a rich source of gene-associated polymorphic markers. Microsatellite-containing cDNAs can be physically mapped and converted to highly informative genetic markers, thus facilitating integration of the human physical, expression and genetic maps.     

A survey of expressed genes in Caenorhabditis elegans.

As an adjunct to the genomic sequencing of Caenorhabditis elegans, we have investigated a representative cDNA library of 1,517 clones. A single sequence read has been obtained from the 5' end of each clone, allowing its characterization with respect to the public databases, and the clones are being localized on the genome map. The result is the identification of about 1,200 of the estimated 15,000 genes of C. elegans. More than 30% of the inferred protein sequences have significant similarity to existing sequences in the databases, providing a route towards in vivo analysis of known genes in the nematode. These clones also provide material for assessing the accuracy of predicted exons and splicing patterns and will lead to a more accurate estimate of the total number of genes in the organism than has hitherto been available.     

Identification of hundreds of conserved and nonconserved human microRNAs

MicroRNAs are noncoding RNAs of approximately 22 nucleotides that suppress translation of target genes by binding to their mRNA and thus have a central role in gene regulation in health and disease. To date, 222 human microRNAs have been identified, 86 by random cloning and sequencing, 43 by computational approaches and the rest as putative microRNAs homologous to microRNAs in other species. To prove our hypothesis that the total number of microRNAs may be much larger and that several have emerged only in primates, we developed an integrative approach combining bioinformatic predictions with microarray analysis and sequence-directed cloning. Here we report the use of this approach to clone and sequence 89 new human microRNAs (nearly doubling the current number of sequenced human microRNAs), 53 of which are not conserved beyond primates. These findings suggest that the total number of human microRNAs is at least 800.     

Mining the human genome using microarrays of open reading frames

To test the hypothesis that the human genome project will uncover many genes not previously discovered by sequencing of expressed sequence tags (ESTs), we designed and produced a set of microarrays using probes based on open reading frames (ORFs) in 350 Mb of finished and draft human sequence. Our approach aims to identify all genes directly from genomic sequence by querying gene expression. We analysed genomic sequence with a suite of ORF prediction programs, selected approximately one ORF per gene, amplified the ORFs from genomic DNA and arrayed the amplicons onto treated glass slides. Of the first 10,000 arrayed ORFs, 31% are completely novel and 29% are similar, but not identical, to sequences in public databases. Approximately one-half of these are expressed in the tissues we queried by microarray. Subsequent verification by other techniques confirmed expression of several of the novel genes. Expressed sequence tags (ESTs) have yielded vast amounts of data, but our results indicate that many genes in the human genome will only be found by genomic sequencing.     

Caenorhabditis elegans expressed sequence tags identify gene families and potential disease gene homologues.

A database containing mapped partial cDNA sequences from Caenorhabditis elegans will provide a ready starting point for identifying nematode homologues of important human genes and determining their functions in C. elegans. A total of 720 expressed sequence tags (ESTs) have been generated from 585 clones randomly selected from a mixed-stage C. elegans cDNA library. Comparison of these ESTs with sequence databases identified 422 new C. elegans genes, of which 317 are not similar to any sequences in the database. Twenty-six new genes have been mapped by YAC clone hybridization. Members of several gene families, including cuticle collagens, GTP-binding proteins, and RNA helicases were discovered. Many of the new genes are similar to known or potential human disease genes, including CFTR and the LDL receptor.     

A transcriptomic analysis of the phylum Nematoda

The phylum Nematoda occupies a huge range of ecological niches, from free-living microbivores to human parasites. We analyzed the genomic biology of the phylum using 265,494 expressed-sequence tag sequences, corresponding to 93,645 putative genes, from 30 species, including 28 parasites. From 35% to 70% of each species' genes had significant similarity to proteins from the model nematode Caenorhabditis elegans. More than half of the putative genes were unique to the phylum, and 23% were unique to the species from which they were derived. We have not yet come close to exhausting the genomic diversity of the phylum. We identified more than 2,600 different known protein domains, some of which had differential abundances between major taxonomic groups of nematodes. We also defined 4,228 nematode-specific protein families from nematode-restricted genes: this class of genes probably underpins species- and higher-level taxonomic disparity. Nematode-specific families are particularly interesting as drug and vaccine targets.     

Genome-wide analysis of DNA copy-number changes using cDNA microarrays.

Gene amplifications and deletions frequently contribute to tumorigenesis. Characterization of these DNA copy-number changes is important for both the basic understanding of cancer and its diagnosis. Comparative genomic hybridization (CGH) was developed to survey DNA copy-number variations across a whole genome. With CGH, differentially labelled test and reference genomic DNAs are co-hybridized to normal metaphase chromosomes, and fluorescence ratios along the length of chromosomes provide a cytogenetic representation of DNA copy-number variation. CGH, however, has a limited ( approximately 20 Mb) mapping resolution, and higher-resolution techniques, such as fluorescence in situ hybridization (FISH), are prohibitively labour-intensive on a genomic scale. Array-based CGH, in which fluorescence ratios at arrayed DNA elements provide a locus-by-locus measure of DNA copy-number variation, represents another means of achieving increased mapping resolution. Published array CGH methods have relied on large genomic clone (for example BAC) array targets and have covered only a small fraction of the human genome. cDNAs representing over 30,000 radiation-hybrid (RH)-mapped human genes provide an alternative and readily available genomic resource for mapping DNA copy-number changes. Although cDNA microarrays have been used extensively to characterize variation in human gene expression, human genomic DNA is a far more complex mixture than the mRNA representation of human cells. Therefore, analysis of DNA copy-number variation using cDNA microarrays would require a sensitivity of detection an order of magnitude greater than has been routinely reported. We describe here a cDNA microarray-based CGH method, and its application to DNA copy-number variation analysis in breast cancer cell lines and tumours. Using this assay, we were able to identify gene amplifications and deletions genome-wide and with high resolution, and compare alterations in DNA copy number and gene expression.     

Exome sequencing identifies recurrent mutations of the splicing factor SF3B1 gene in chronic lymphocytic leukemia

Here we perform whole-exome sequencing of samples from 105 individuals with chronic lymphocytic leukemia (CLL), the most frequent leukemia in adults in Western countries. We found 1,246 somatic mutations potentially affecting gene function and identified 78 genes with predicted functional alterations in more than one tumor sample. Among these genes, SF3B1, encoding a subunit of the spliceosomal U2 small nuclear ribonucleoprotein (snRNP), is somatically mutated in 9.7% of affected individuals. Further analysis in 279 individuals with CLL showed that SF3B1 mutations were associated with faster disease progression and poor overall survival. This work provides the first comprehensive catalog of somatic mutations in CLL with relevant clinical correlates and defines a large set of new genes that may drive the development of this common form of leukemia. The results reinforce the idea that targeting several well-known genetic pathways, including mRNA splicing, could be useful in the treatment of CLL and other malignancies.     

Resequencing of 200 human exomes identifies an excess of low-frequency non-synonymous coding variants

Targeted capture combined with massively parallel exome sequencing is a promising approach to identify genetic variants implicated in human traits. We report exome sequencing of 200 individuals from Denmark with targeted capture of 18,654 coding genes and sequence coverage of each individual exome at an average depth of 12-fold. On average, about 95% of the target regions were covered by at least one read. We identified 121,870 SNPs in the sample population, including 53,081 coding SNPs (cSNPs). Using a statistical method for SNP calling and an estimation of allelic frequencies based on our population data, we derived the allele frequency spectrum of cSNPs with a minor allele frequency greater than 0.02. We identified a 1.8-fold excess of deleterious, non-syonomyous cSNPs over synonymous cSNPs in the low-frequency range (minor allele frequencies between 2% and 5%). This excess was more pronounced for X-linked SNPs, suggesting that deleterious substitutions are primarily recessive.     

Enzymatic production of RNAi libraries from cDNAs

RNA interference (RNAi) induced by small interfering (siRNA) or short hairpin RNA (shRNA) is an important research approach in mammalian genetics. Here we describe a technology called enzymatic production of RNAi library (EPRIL) by which cDNAs are converted by a sequence of enzymatic treatments into an RNAi library consisting of a vast array of different shRNA expression constructs. We applied EPRIL to a single cDNA source and prepared an RNAi library consisting of shRNA constructs with various RNAi efficiencies. High-throughput screening allowed us to rapidly identify the best shRNA constructs from the library. We also describe a new selection scheme using the thymidine kinase gene for obtaining efficient shRNA constructs. Furthermore, we show that EPRIL can be applied to constructing an RNAi library from a cDNA library, providing a basis for future whole-genome phenotypic screening of genes.     

Diversity of microRNAs in human and chimpanzee brain

We used massively parallel sequencing to compare the microRNA (miRNA) content of human and chimpanzee brains, and we identified 447 new miRNA genes. Many of the new miRNAs are not conserved beyond primates, indicating their recent origin, and some miRNAs seem species specific, whereas others are expanded in one species through duplication events. These data suggest that evolution of miRNAs is an ongoing process and that along with ancient, highly conserved miRNAs, there are a number of emerging miRNAs.     

Evidence of en bloc duplication in vertebrate genomes

It has been 30 years since it was first proposed that the vertebrate genome evolved through several rounds of genome-wide duplications (polyploidizations). Despite rapid advances in genetics, including sequencing of the complete genomes of several divergent species, this hypothesis has not been tested rigorously and is still a matter of debate. If polyploidizations occurred during chordate evolution, there should be a network of paralogous regions in the present-day jawed vertebrate (Gnathostomata) genomes. Here we present an investigation of the major histocompatibility complex (MHC) paralogous regions, which we accomplished by characterizing the corresponding region in amphioxus by identifying nine anchor genes and sequencing both the anchor genes and the regions that flank them (a total of 400 kb). Phylogenetic analysis of 31 genes (including the anchor genes) in these regions shows that duplications occurred after the divergence of cephalochordates and vertebrates but before the Gnathostomata radiation. The distribution of human and amphioxus orthologs in their respective genomes and the relationship between these distributions support the en bloc duplication events. Our analysis represents the first step towards demonstrating that the human ancestral genome has undergone polyploidization. Moreover, reconstruction of the pre-duplicated region indicates that one of the duplicated regions retains the ancestral organization.