New insights into immune genes and other expanded gene families of the house fly, Musca domestica, from an improved whole genome sequence.

The house fly, Musca domestica, is a pest of livestock, transmits pathogens of human diseases, and is a model organism in multiple biological research areas. The first house fly genome assembly was published in 2014 and has been of tremendous use to the community of house fly biologists, but that genome is discontiguous and incomplete by contemporary standards. To improve the house fly reference genome, we sequenced, assembled, and annotated the house fly genome using improved techniques and technologies that were not available at the time of the original genome sequencing project. The new genome assembly is substantially more contiguous and complete than the previous genome. The new genome assembly has a scaffold N50 of 12.46 Mb, which is a 50-fold improvement over the previous assembly. In addition, the new genome assembly is within 1% of the estimated genome size based on flow cytometry, whereas the previous assembly was missing nearly one-third of the predicted genome sequence. The improved genome assembly has much more contiguous scaffolds containing large gene families. To provide an example of the benefit of the new genome, we used it to investigate tandemly arrayed immune gene families. The new contiguous assembly of these loci provides a clearer picture of the regulation of the expression of immune genes, and it leads to new insights into the selection pressures that shape their evolution.

Genome wide natural variation of H3K27me3 selectively marks genes predicted to be important for cell differentiation in Phaeodactylum tricornutum.

In multicellular organisms, Polycomb Repressive Complex2 (PRC2) is known to deposit tri-methylation of lysine 27 of histone H3 (H3K27me3) to establish and maintain gene silencing, critical for developmentally regulated processes. The PRC2 complex is absent in both widely studied model yeasts, which initially suggested that PRC2 arose with the emergence of multicellularity. However, its discovery in several unicellular species including microalgae questions its role in unicellular eukaryotes. Here, we use Phaeodactylum tricornutum enhancer of zeste E(z) knockouts and show that P. tricornutum E(z) is responsible for di- and tri-methylation of lysine 27 of histone H3. H3K27me3 depletion abolishes cell morphology in P. tricornutum providing evidence for its role in cell differentiation. Genome-wide profiling of H3K27me3 in fusiform and triradiate cells further revealed genes that may specify cell identity. These results suggest a role for PRC2 and its associated mark in cell differentiation in unicellular species, and highlight their ancestral function in a broader evolutionary context than currently is appreciated.

Genome-wide analysis of allele-specific expression of genes in the model diatom Phaeodactylum tricornutum.

Recent advances in next generation sequencing technologies have allowed the discovery of widespread autosomal allele-specific expression (aASE) in mammals and plants with potential phenotypic effects. Extensive numbers of genes with allele-specific expression have been described in the diatom Fragilariopsis cylindrus in association with adaptation to external cues, as well as in Fistulifera solaris in the context of natural hybridization. However, the role of aASE and its extent in diatoms remain elusive. In this study, we investigate allele-specific expression in the model diatom Phaeodactylum tricornutum by the re-analysis of previously published whole genome RNA sequencing data and polymorphism calling. We found that 22% of P. tricornutum genes show moderate bias in allelic expression while 1% show nearly complete monoallelic expression. Biallelic expression associates with genes encoding components of protein metabolism while moderately biased genes associate with functions in catabolism and protein transport. We validated candidate genes by pyrosequencing and found that moderate biases in allelic expression were less stable than monoallelically expressed genes that showed consistent bias upon experimental validations at the population level and in subcloning experiments. Our approach provides the basis for the analysis of aASE in P. tricornutum and could be routinely implemented to test for variations in allele expression under different environmental conditions.

A genomics approach reveals the global genetic polymorphism, structure, and functional diversity of ten accessions of the marine model diatom Phaeodactylum tricornutum.

Diatoms emerged in the Mesozoic period and presently constitute one of the main primary producers in the world's ocean and are of a major economic importance. In the current study, using whole genome sequencing of ten accessions of the model diatom Phaeodactylum tricornutum, sampled at broad geospatial and temporal scales, we draw a comprehensive landscape of the genomic diversity within the species. We describe strong genetic subdivisions of the accessions into four genetic clades (A-D) with constituent populations of each clade possessing a conserved genetic and functional makeup, likely a consequence of the limited dispersal of P. tricornutum in the open ocean. We further suggest dominance of asexual reproduction across all the populations, as implied by high linkage disequilibrium. Finally, we show limited yet compelling signatures of genetic and functional convergence inducing changes in the selection pressure on many genes and metabolic pathways. We propose these findings to have significant implications for understanding the genetic structure of diatom populations in nature and provide a framework to assess the genomic underpinnings of their ecological success and impact on aquatic ecosystems where they play a major role. Our work provides valuable resources for functional genomics and for exploiting the biotechnological potential of this model diatom species.

Integrative analysis of large scale transcriptome data draws a comprehensive landscape of Phaeodactylum tricornutum genome and evolutionary origin of diatoms.

Diatoms are one of the most successful and ecologically important groups of eukaryotic phytoplankton in the modern ocean. Deciphering their genomes is a key step towards better understanding of their biological innovations, evolutionary origins, and ecological underpinnings. Here, we have used 90 RNA-Seq datasets from different growth conditions combined with published expressed sequence tags and protein sequences from multiple taxa to explore the genome of the model diatom Phaeodactylum tricornutum, and introduce 1,489 novel genes. The new annotation additionally permitted the discovery of extensive alternative splicing in diatoms, including intron retention and exon skipping, which increase the diversity of transcripts generated in changing environments. In addition, we have used up-to-date reference sequence libraries to dissect the taxonomic origins of diatom genes. We show that the P. tricornutum genome is enriched in lineage-specific genes, with up to 47% of the gene models present only possessing orthologues in other stramenopile groups. Finally, we have performed a comprehensive de novo annotation of repetitive elements showing novel classes of transposable elements such as SINE, MITE and TRIM/LARD. This work provides a solid foundation for future studies of diatom gene function, evolution and ecology.

Recent progress in diatom genomics and epigenomics.

Diatoms are one of the most diverse and successful groups of phytoplankton at the base of the food chain, sustaining life in the ocean and performing vital biogeochemical functions. The last fifteen years have witnessed the comprehensive analysis of several diatom genomes, revealing that they bear traces of their endosymbiotic origins from algal and heterotrophic ancestors, as well as significant gene transfer from bacteria. Their chimeric genomes are further regulated by a range of chromatin-based processes that are characteristic of both plant and animal genomes. We discuss the conservation of gene regulatory mechanisms in diatoms and propose that epigenetic processes may have a significant role in mediating responses to a highly dynamic and unpredictable environment in these organisms.

PhytoCRISP-Ex: a web-based and stand-alone application to find specific target sequences for CRISPR/CAS editing.

BACKGROUND: With the emerging interest in phytoplankton research, the need to establish genetic tools for the functional characterization of genes is indispensable. The CRISPR/Cas9 system is now well recognized as an efficient and accurate reverse genetic tool for genome editing. Several computational tools have been published allowing researchers to find candidate target sequences for the engineering of the CRISPR vectors, while searching possible off-targets for the predicted candidates. These tools provide built-in genome databases of common model organisms that are used for CRISPR target prediction. Although their predictions are highly sensitive, the applicability to non-model genomes, most notably protists, makes their design inadequate. This motivated us to design a new CRISPR target finding tool, PhytoCRISP-Ex. Our software offers CRIPSR target predictions using an extended list of phytoplankton genomes and also delivers a user-friendly standalone application that can be used for any genome. RESULTS: The software attempts to integrate, for the first time, most available phytoplankton genomes information and provide a web-based platform for Cas9 target prediction within them with high sensitivity. By offering a standalone version, PhytoCRISP-Ex maintains an independence to be used with any organism and widens its applicability in high throughput pipelines. PhytoCRISP-Ex out pars all the existing tools by computing the availability of restriction sites over the most probable Cas9 cleavage sites, which can be ideal for mutant screens. CONCLUSIONS: PhytoCRISP-Ex is a simple, fast and accurate web interface with 13 pre-indexed and presently updating phytoplankton genomes. The software was also designed as a UNIX-based standalone application that allows the user to search for target sequences in the genomes of a variety of other species.

An integrative analysis of post-translational histone modifications in the marine diatom Phaeodactylum tricornutum.

BACKGROUND: Nucleosomes are the building blocks of chromatin where gene regulation takes place. Chromatin landscapes have been profiled for several species, providing insights into the fundamental mechanisms of chromatin-mediated transcriptional regulation of gene expression. However, knowledge is missing for several major and deep-branching eukaryotic groups, such as the Stramenopiles, which include the diatoms. Diatoms are highly diverse and ubiquitous species of phytoplankton that play a key role in global biogeochemical cycles. Dissecting chromatin-mediated regulation of genes in diatoms will help understand the ecological success of these organisms in contemporary oceans. RESULTS: Here, we use high resolution mass spectrometry to identify a full repertoire of post-translational modifications on histones of the marine diatom Phaeodactylum tricornutum, including eight novel modifications. We map five histone marks coupled with expression data and show that P. tricornutum displays both unique and broadly conserved chromatin features, reflecting the chimeric nature of its genome. Combinatorial analysis of histone marks and DNA methylation demonstrates the presence of an epigenetic code defining activating or repressive chromatin states. We further profile three specific histone marks under conditions of nitrate depletion and show that the histone code is dynamic and targets specific sets of genes. CONCLUSIONS: This study is the first genome-wide characterization of the histone code from a stramenopile and a marine phytoplankton. The work represents an important initial step for understanding the evolutionary history of chromatin and how epigenetic modifications affect gene expression in response to environmental cues in marine environments.

Whole genome sequencing of an ethnic Pathan (Pakhtun) from the north-west of Pakistan.

BACKGROUND: Pakistan covers a key geographic area in human history, being both part of the Indus River region that acted as one of the cradles of civilization and as a link between Western Eurasia and Eastern Asia. This region is inhabited by a number of distinct ethnic groups, the largest being the Punjabi, Pathan (Pakhtuns), Sindhi, and Baloch. RESULTS: We analyzed the first ethnic male Pathan genome by sequencing it to 29.7-fold coverage using the Illumina HiSeq2000 platform. A total of 3.8 million single nucleotide variations (SNVs) and 0.5 million small indels were identified by comparing with the human reference genome. Among the SNVs, 129,441 were novel, and 10,315 nonsynonymous SNVs were found in 5,344 genes. SNVs were annotated for health consequences and high risk diseases, as well as possible influences on drug efficacy. We confirmed that the Pathan genome presented here is representative of this ethnic group by comparing it to a panel of Central Asians from the HGDP-CEPH panels typed for ~650 k SNPs. The mtDNA (H2) and Y haplogroup (L1) of this individual were also typical of his geographic region of origin. Finally, we reconstruct the demographic history by PSMC, which highlights a recent increase in effective population size compatible with admixture between European and Asian lineages expected in this geographic region. CONCLUSIONS: We present a whole-genome sequence and analyses of an ethnic Pathan from the north-west province of Pakistan. It is a useful resource to understand genetic variation and human migration across the whole Asian continent.

GFF-Ex: a genome feature extraction package.

BACKGROUND: Genomic features of whole genome sequences emerging from various sequencing and annotation projects are represented and stored in several formats. Amongst these formats, the GFF (Generic/General Feature Format) has emerged as a widely accepted, portable and successfully used flat file format for genome annotation storage. With an increasing interest in genome annotation projects and secondary and meta-analysis, there is a need for efficient tools to extract sequences of interests from GFF files. FINDINGS: We have developed GFF-Ex to automate feature-based extraction of sequences from a GFF file. In addition to automated sequence extraction of the features described within a feature file, GFF-Ex also assigns boundaries for the features (introns, intergenic, regions upstream to genes), which are not explicitly specified in the GFF format, and exports the corresponding primary sequence information into predefined feature specific output files. GFF-Ex package consists of several UNIX Shell and PERL scripts. CONCLUSIONS: Compared to other available GFF parsers, GFF-Ex is a simpler tool, which permits sequence retrieval based on additional inferred features. GFF-Ex can also be integrated with any genome annotation or analysis pipeline. GFF-Ex is freely available at http://bioinfo.icgeb.res.in/gff.