Cancer Moonshot Data and Technology Team: Enabling a National Learning Healthcare System for Cancer to Unleash the Power of Data.

The Cancer Moonshot emphasizes the need to learn from the experiences of cancer patients to positively impact their outcomes, experiences, and qualities of life. To realize this vision, there has been a concerted effort to identify the fundamental building blocks required to establish a National Learning Healthcare System for Cancer, such that relevant data on all cancer patients is accessible, shareable, and contributing to the current state of knowledge of cancer care and outcomes.

Skewed oligomers and origins of replication.

The putative origin of replication in prokaryotic genomes can be located by a new method that finds short oligomers whose orientation is preferentially skewed around the origin. The skewed oligomer method is shown to work for all bacterial genomes and one of three archaeal genomes sequences to date, confirming known or predicted origins in most cases and in three cases (H. pylori, M. thermoautotrophicum, and Synechocystis sp.), suggesting origins that were previously unknown. In many cases, the presence of conserved genes and nucleotide motifs confirms the predictions. An algorithm for finding these skewed seven-base and eight-base sequences is described, along with a method for combining evidence from multiple skewed oligomers to accurately locate the replication origin. Possible explanations for the phenomenon of skewed oligomers are discussed. Explanations are presented for why some bacterial genomes contain hundreds of highly skewed oligomers, whereas others contain only a handful.

Characterization of the myoglobin and its coding gene of the mollusc Biomphalaria glabrata.

A cDNA clone isolated from a Biomphalaria glabrata (Mollusca, Gastropoda) neural cDNA library was identified as encoding a myoglobin-like protein of 148 amino acids with a single domain and a calculated mass of 16,049.29. Alignment with globin sequences with known tertiary structure confirms its overall globin nature. The expressed myoglobin was identified in the radular muscle and isolated. Oxygen equilibrium measurements on the protein reveal a high oxygen affinity. Val-B10 and Gln-E7, important residues for the determination of the oxygen affinity, are strikingly different from the standard molluscan pattern (Conti, E., Moser, C., Rizzi, M., Mattevi, A., Lionetti, C., Coda, A., Ascenzi, P., Brunori, M., Bolognesi, M. (1993) J. Mol. Biol. 233, 498-508). The single gene encoding the globin chain is interrupted by three introns at positions A3.2, B12.2, and G7.0. Comparison with other nonvertebrate globin genes reveals on the one hand conservation (B12.2 and G7.0) and on the other hand variability of the insertion positions (A3.2). The Biomphalaria myoglobin sequence was used together with all other molluscan globin sequences available to assess the origin and phylogeny of the phylum. Our results confirm the doubts raised about monophyletic origin of the Mollusca, which was first observed using SSU rRNA as a molecular marker.

Genomic sequence of a Lyme disease spirochaete, Borrelia burgdorferi.

The genome of the bacterium Borrelia burgdorferi B31, the aetiologic agent of Lyme disease, contains a linear chromosome of 910,725 base pairs and at least 17 linear and circular plasmids with a combined size of more than 533,000 base pairs. The chromosome contains 853 genes encoding a basic set of proteins for DNA replication, transcription, translation, solute transport and energy metabolism, but, like Mycoplasma genitalium, it contains no genes for cellular biosynthetic reactions. Because B. burgdorferi and M. genitalium are distantly related eubacteria, we suggest that their limited metabolic capacities reflect convergent evolution by gene loss from more metabolically competent progenitors. Of 430 genes on 11 plasmids, most have no known biological function; 39% of plasmid genes are paralogues that form 47 gene families. The biological significance of the multiple plasmid-encoded genes is not clear, although they may be involved in antigenic variation or immune evasion.

The complete genome sequence of the hyperthermophilic, sulphate-reducing archaeon Archaeoglobus fulgidus.

Archaeoglobus fulgidus is the first sulphur-metabolizing organism to have its genome sequence determined. Its genome of 2,178,400 base pairs contains 2,436 open reading frames (ORFs). The information processing systems and the biosynthetic pathways for essential components (nucleotides, amino acids and cofactors) have extensive correlation with their counterparts in the archaeon Methanococcus jannaschii. The genomes of these two Archaea indicate dramatic differences in the way these organisms sense their environment, perform regulatory and transport functions, and gain energy. In contrast to M. jannaschii, A. fulgidus has fewer restriction-modification systems, and none of its genes appears to contain inteins. A quarter (651 ORFs) of the A. fulgidus genome encodes functionally uncharacterized yet conserved proteins, two-thirds of which are shared with M. jannaschii (428 ORFs). Another quarter of the genome encodes new proteins indicating substantial archaeal gene diversity.

A tool for analyzing and annotating genomic sequences.

We describe a tool for analyzing and annotating large genomic sequences containing introns. The analysis and annotation tool (AAT) includes two sets of programs, one for comparing the query sequence with a protein database and the other for comparing the query with a cDNA database. Each set contains a fast database search program and a rigorous alignment program. The database search program quickly identifies regions of the query sequence that are similar to a database sequence. Then the alignment program constructs an optimal alignment for each region and the database sequence. The alignment program also reports the coordinates of exons in the query sequence. Pairwise alignments of the query sequence with protein and cDNA database sequences are combined into multiple sequence alignments, which provide a view of all protein and cDNA sequences matching a query region. On a data set of 570 DNA sequences, AAT identified 94% of coding nucleotides correctly and 74% of exons exactly. Results of analyzing a human BAC sequence with the AAT tool are also presented. The AAT tool reduces the labor-intensive work of locating the exons of the query sequence and improves the process of defining intron-exon boundaries by using the wealth of available protein and cDNA data.

The complete genome sequence of the gastric pathogen Helicobacter pylori.

Helicobacter pylori, strain 26695, has a circular genome of 1,667,867 base pairs and 1,590 predicted coding sequences. Sequence analysis indicates that H. pylori has well-developed systems for motility, for scavenging iron, and for DNA restriction and modification. Many putative adhesins, lipoproteins and other outer membrane proteins were identified, underscoring the potential complexity of host-pathogen interaction. Based on the large number of sequence-related genes encoding outer membrane proteins and the presence of homopolymeric tracts and dinucleotide repeats in coding sequences, H. pylori, like several other mucosal pathogens, probably uses recombination and slipped-strand mispairing within repeats as mechanisms for antigenic variation and adaptive evolution. Consistent with its restricted niche, H. pylori has a few regulatory networks, and a limited metabolic repertoire and biosynthetic capacity. Its survival in acid conditions depends, in part, on its ability to establish a positive inside-membrane potential in low pH.

The construction of Arabidopsis expressed sequence tag assemblies. A new resource to facilitate gene identification.

The generation of large numbers of partial cDNA sequences, or expressed sequence tags (ESTs), has provided a method with which to sample a large number of genes from an organism. More than 25,000 Arabidopsis thaliana ESTs have been deposited in public databases, producing the largest collection of ESTs for any plant species. We describe here the application of a method of reducing redundancy and increasing information content in this collection by grouping overlapping ESTs representing the same gene into a "contig" or assembly. The increased information content of these assemblies allows more putative identifications to be assigned based on the results of similarity searches with nucleotide and protein databases. The results of this analysis indicate that sequence information is available for approximately 12,600 nonoverlapping ESTs from Arabidopsis. Comparison of the assemblies with 953 Arabidopsis coding sequences indicates that up to 57% of all Arabidopsis genes are represented by an EST. Clustering analysis of these sequences suggests that between 300 and 700 gene families are represented by between 700 and 2000 sequences in the EST database. A database of the assembled sequences, their putative identifications, and cellular roles is available through the World Wide Web.

Complete genome sequence of the methanogenic archaeon, Methanococcus jannaschii.

The complete 1.66-megabase pair genome sequence of an autotrophic archaeon, Methanococcus jannaschii, and its 58- and 16-kilobase pair extrachromosomal elements have been determined by whole-genome random sequencing. A total of 1738 predicted protein-coding genes were identified; however, only a minority of these (38 percent) could be assigned a putative cellular role with high confidence. Although the majority of genes related to energy production, cell division, and metabolism in M. jannaschii are most similar to those found in Bacteria, most of the genes involved in transcription, translation, and replication in M. jannaschii are more similar to those found in Eukaryotes.

The minimal gene complement of Mycoplasma genitalium.

The complete nucleotide sequence (580,070 base pairs) of the Mycoplasma genitalium genome, the smallest known genome of any free-living organism, has been determined by whole-genome random sequencing and assembly. A total of only 470 predicted coding regions were identified that include genes required for DNA replication, transcription and translation, DNA repair, cellular transport, and energy metabolism. Comparison of this genome to that of Haemophilus influenzae suggests that differences in genome content are reflected as profound differences in physiology and metabolic capacity between these two organisms.

Initial assessment of human gene diversity and expression patterns based upon 83 million nucleotides of cDNA sequence.

In an effort to identify new genes and analyse their expression patterns, 174,472 partial complementary DNA sequences (expressed sequence tags (ESTs)), totalling more than 52 million nucleotides of human DNA sequence, have been generated from 300 cDNA libraries constructed from 37 distinct organs and tissues. These ESTs have been combined with an additional 118,406 ESTs from the database dbEST, for a total of 83 million nucleotides, and treated as a shotgun sequence assembly project. The assembly process yielded 29,599 distinct tentative human consensus (THC) sequences and 58,384 non-overlapping ESTs. Of these 87,983 distinct sequences, 10,214 further characterize previously known genes based on statistically significant similarity to sequences in the available databases; the remainder identify previously unknown genes. Thirty tissues were sampled by over 1,000 ESTs each; only eight genes were matched by ESTs from all 30 tissues, and 227 genes were represented in 20 or more of the tissues sampled with more than 1,000 ESTs. Approximately 40% of identified human genes appear to be associated with basic energy metabolism, cell structure, homeostasis and cell division, 22% with RNA and protein synthesis and processing, and 12% with cell signalling and communication.

Comparative expressed-sequence-tag analysis of differential gene expression profiles in PC-12 cells before and after nerve growth factor treatment.

Nerve growth factor-induced differentiation of adrenal chromaffin PC-12 cells to a neuronal phenotype involves alterations in gene expression and represents a model system to study neuronal differentiation. We have used the expressed-sequence-tag approach to identify approximately 600 differentially expressed mRNAs in untreated and nerve growth factor-treated PC-12 cells that encode proteins with diverse structural and biochemical functions. Many of these mRNAs encode proteins belonging to cellular pathways not previously known to be regulated by nerve growth factor. Comparative expressed-sequence-tag analysis provides a basis for surveying global changes in gene-expression patterns in response to biological signals at an unprecedented scale, is a powerful tool for identifying potential interactions between different cellular pathways, and allows the gene-expression profiles of individual genes belonging to a particular pathway to be followed.

Whole-genome random sequencing and assembly of Haemophilus influenzae Rd.

An approach for genome analysis based on sequencing and assembly of unselected pieces of DNA from the whole chromosome has been applied to obtain the complete nucleotide sequence (1,830,137 base pairs) of the genome from the bacterium Haemophilus influenzae Rd. This approach eliminates the need for initial mapping efforts and is therefore applicable to the vast array of microbial species for which genome maps are unavailable. The H. influenzae Rd genome sequence (Genome Sequence DataBase accession number L42023) represents the only complete genome sequence from a free-living organism.

Analysis of the complete genome of smallpox variola major virus strain Bangladesh-1975.

We analyzed the 186,102 base pairs (bp) that constitute the entire DNA genome of a highly virulent variola virus isolated from Bangladesh in 1975. The linear, double-stranded molecule has relatively small (725 bp) inverted terminal repeat (ITR) sequences containing three 69-bp direct repeat elements, a 54-bp partial repeat element, and a 105-base telomeric end-loop that can be maximally base-paired to contain 17 mismatches. Proximal to the right-end ITR sequences are another seven 69-bp elements and a 53- and a 27-bp partial element. Sequence analysis showed 187 closely spaced open reading frames specifying putative major proteins containing > or = 65 amino acids. Most of the virus proteins correspond to proteins in current databases, including 150 proteins that have > 90% identity to major gene products encoded by vaccinia virus, the smallpox vaccine. Variola virus has a group of proteins that are truncated compared with vaccinia virus counterparts and a smaller group of proteins that are elongated. The terminal regions encode several novel proteins and variants of other poxvirus proteins that potentially augment variola virus transmissibility and virulence for its only natural host, humans.