Anuj Srivastava

Assemblathon 1: A competitive assessment of de novo short read assembly methods

Low-cost short read sequencing technology has revolutionized genomics, though it is only just becoming practical for the high-quality de novo assembly of a novel large genome. We describe the Assemblathon 1 competition, which aimed to comprehensively assess the state of the art in de novo assembly methods when applied to current sequencing technologies. In a collaborative effort, teams were asked to assemble a simulated Illumina HiSeq data set of an unknown, simulated diploid genome. A total of 41 assemblies from 17 different groups were received. Novel haplotype aware assessments of coverage, contiguity, structure, base calling, and copy number were made. We establish that within this benchmark: (1) It is possible to assemble the genome to a high level of coverage and accuracy, and that (2) large differences exist between the assemblies, suggesting room for further improvements in current methods. The simulated benchmark, including the correct answer, the assemblies, and the code that was used to evaluate the assemblies is now public and freely available from http://www.assemblathon.org/.

Transcriptome Sequencing and Annotation for the Jamaican Fruit Bat (Artibeus jamaicensis)

The Jamaican fruit bat (Artibeus jamaicensis) is one of the most common bats in the tropical Americas. It is thought to be a potential reservoir host of Tacaribe virus, an arenavirus closely related to the South American hemorrhagic fever viruses. We performed transcriptome sequencing and annotation from lung, kidney and spleen tissues using 454 and Illumina platforms to develop this species as an animal model. More than 100,000 contigs were assembled, with 25,000 genes that were functionally annotated. Of the remaining unannotated contigs, 80% were found within bat genomes or transcriptomes. Annotated genes are involved in a broad range of activities ranging from cellular metabolism to genome regulation through ncRNAs. Reciprocal BLAST best hits yielded 8,785 sequences that are orthologous to mouse, rat, cattle, horse and human. Species tree analysis of sequences from 2,378 loci was used to achieve 95% bootstrap support for the placement of bat as sister to the clade containing horse, dog, and cattle. Through substitution rate estimation between bat and human, 32 genes were identified with evidence for positive selection. We also identified 466 immune-related genes, which may be useful for studying Tacaribe virus infection of this species. The Jamaican fruit bat transcriptome dataset is a resource that should provide additional candidate markers for studying bat evolution and ecology, and tools for analysis of the host response and pathology of disease.

Transcriptome Analysis of Sarracenia, an Insectivorous Plant

Sarracenia species (pitcher plants) are carnivorous plants which obtain a portion of their nutrients from insects captured in the pitchers. To investigate these plants, we sequenced the transcriptome of two species, Sarracenia psittacina and Sarracenia purpurea, using Roche 454 pyrosequencing technology. We obtained 46 275 and 36 681 contigs by de novo assembly methods for S. psittacina and S. purpurea, respectively, and further identified 16 163 orthologous contigs between them. Estimation of synonymous substitution rates between orthologous and paralogous contigs indicates the events of genome duplication and speciation within the Sarracenia genus both occurred ∼2 million years ago. The ratios of synonymous and non-synonymous substitution rates indicated that 491 contigs have been under positive selection (Ka/Ks > 1). Significant proportions of these contigs were involved in functions related to binding activity. We also found that the greatest sequence similarity for both of these species was to Vitis vinifera, which is most consistent with a non-current classification of the order Ericales as an asterid. This study has provided new insights into pitcher plants and will contribute greatly to future research on this genus and its distinctive ecological adaptations.

Exome sequencing reveals pathogenic mutations in 91 strains of mice with Mendelian disorders

Spontaneously arising mouse mutations have served as the foundation for understanding gene function for more than 100 years. We have used exome sequencing in an effort to identify the causative mutations for 172 distinct, spontaneously arising mouse models of Mendelian disorders, including a broad range of clinically relevant phenotypes. To analyze the resulting data, we developed an analytics pipeline that is optimized for mouse exome data and a variation database that allows for reproducible, user-defined data mining as well as nomination of mutation candidates through knowledge-based integration of sample and variant data. Using these new tools, putative pathogenic mutations were identified for 91 (53%) of the strains in our study. Despite the increased power offered by potentially unlimited pedigrees and controlled breeding, about half of our exome cases remained unsolved. Using a combination of manual analyses of exome alignments and whole-genome sequencing, we provide evidence that a large fraction of unsolved exome cases have underlying structural mutations. This result directly informs efforts to investigate the similar proportion of apparently Mendelian human phenotypes that are recalcitrant to exome sequencing.

Development and validation of the JAX Cancer Treatment Profile™ for detection of clinically actionable mutations in solid tumors

BACKGROUND The continued development of targeted therapeutics for cancer treatment has required the concomitant development of more expansive methods for the molecular profiling of the patients tumor. We describe the validation of the JAX Cancer Treatment Profile™ (JAX-CTP™), a next generation sequencing (NGS)-based molecular diagnostic assay that detects actionable mutations in solid tumors to inform the selection of targeted therapeutics for cancer treatment. METHODS NGS libraries are generated from DNA extracted from formalin fixed paraffin embedded tumors. Using hybrid capture, the genes of interest are enriched and sequenced on the Illumina HiSeq 2500 or MiSeq sequencers followed by variant detection and functional and clinical annotation for the generation of a clinical report. RESULTS The JAX-CTP™ detects actionable variants, in the form of single nucleotide variations and small insertions and deletions (≤50 bp) in 190 genes in specimens with a neoplastic cell content of ≥10%. The JAX-CTP™ is also validated for the detection of clinically actionable gene amplifications. CONCLUSIONS There is a lack of consensus in the molecular diagnostics field on the best method for the validation of NGS-based assays in oncology, thus the importance of communicating methods, as contained in this report. The growing number of targeted therapeutics and the complexity of the tumor genome necessitate continued development and refinement of advanced assays for tumor profiling to enable precision cancer treatment.

Transcriptome Analysis of a North American Songbird, Melospiza melodia

An effective way to understand the genomics of divergence in non-model organisms is to use the transcriptome to identify genes associated with divergence. We examine the transcriptome of the song sparrow (Melospiza melodia) and contrast it with the avian models zebra finch (Taeniopygia guttata) and chicken (Gallus gallus). We aimed to (i) obtain a functional annotation of a substantial portion of the song sparrow transcriptome; (ii) compare transcript divergence; (iii) efficiently characterize single nucleotide polymorphism/indel markers possibly fixed between song sparrow subspecies; and (iv) identify the most common set of transcripts in birds using the zebra finch as a reference. Using two individuals from each of three populations, whole-body mRNA was normalized and sequenced (110Mb total). The assembly yielded 38 539 contigs [N50 (the length–weighted median) = 482 bp]; 4574 were orthologous to both model genomes and 3680 are functionally annotated. This low-coverage scan of the song sparrow transcriptome revealed 29 982 SNPs/indels, 1402 fixed between populations and subspecies. Referencing zebra finch and chicken, we identified 43 and 5 fast-evolving genes, respectively. We also identified the most common set of transcripts present in birds with respect to zebra finch. This study provides new insight into songbird transcriptomes, and candidate markers identified here may help research in songbirds (oscine Passeriformes), a frequently studied group.

AIMIE: a web-based environment for detection and interpretation of significant sequence motifs in prokaryotic genomes

MOTIVATION Genomes contain biologically significant information that extends beyond that encoded in genes. Some of this information relates to various short dispersed repeats distributed throughout the genome. The goal of this work was to combine tools for detection of statistically significant dispersed repeats in DNA sequences with tools to aid development of hypotheses regarding their possible physiological functions in an easy-to-use web-based environment. RESULTS Ab Initio Motif Identification Environment (AIMIE) was designed to facilitate investigations of dispersed sequence motifs in prokaryotic genomes. We used AIMIE to analyze the Escherichia coli and Haemophilus influenzae genomes in order to demonstrate the utility of the new environment. AIMIE detected repeated extragenic palindrome (REP) elements, CRISPR repeats, uptake signal sequences, intergenic dyad sequences and several other over-represented sequence motifs. Distributional patterns of these motifs were analyzed using the tools included in AIMIE. AVAILABILITY AIMIE and the related software can be accessed at our web site http://www.cmbl.uga.edu/software.html.

Mutational patterns in RNA secondary structure evolution examined in three RNA families.

The goal of this work was to study mutational patterns in the evolution of RNA secondary structure. We analyzed bacterial tmRNA, RNaseP and eukaryotic telomerase RNA secondary structures, mapping structural variability onto phylogenetic trees constructed primarily from rRNA sequences. We found that secondary structures evolve both by whole stem insertion/deletion, and by mutations that create or disrupt stem base pairing. We analyzed the evolution of stem lengths and constructed substitution matrices describing the changes responsible for the variation in the RNA stem length. In addition, we used principal component analysis of the stem length data to determine the most variable stems in different families of RNA. This data provides new insights into the evolution of RNA secondary structures and patterns of variation in the lengths of double helical regions of RNA molecules. Our findings will facilitate design of improved mutational models for RNA structure evolution.

Patterns of chromatin-modifications discriminate different genomic features in Arabidopsis

Motivation: Dynamic regulation and packaging of genetic information is achieved by the organization of DNA into chromatin. Nucleosomal core histones, which form the basic repeating unit of chromatin, are subject to various post-translational modifications such as acetylation, methylation, phosphorylation and ubiquitinylation. These modifications have effects on chromatin structure and, along with DNA methylation, regulate gene transcription. The goal of this study was to determine if patterns in modifications were related to different categories of genomic features, and, if so, if the patterns had predictive value.