MiRAGDB: A Knowledgebase of RAG Regulators.

RAG1 and RAG2 genes generate diversity in immunoglobulin and TCR genes by initiating the process of V-D-J recombination. RAGs recognize specific sequences (heptamer-nonamer) to generate a diversity of immunoglobulins. RAG expression is limited to early B and T cell developmental stages. Aberrant expression of RAG can lead to double strand breaks and translocations as observed in leukemia and lymphoma. The expression of RAG is tightly regulated at the transcriptional and posttranscriptional levels. MicroRNAs (miRNAs) are small non-coding RNAs that are involved in the post-transcriptional regulation of gene expression. This study aimed to identify and catalog RAG regulation by miRNA during normal development and cancer. NGS data from normal B-cell and T-cell developmental stages and blood cancer samples have been analyzed for the expression of miRNAs against RAG1 (1,173 against human RAG1 and 749 against mouse RAG1). The analyzed data has been organized to retrieve the miRNA and mRNA expression of various RAG regulators (10 transcription factors and interacting partners) in normal and diseased states. The database allows users to navigate through the human and mouse RAG regulators, visualize and plot expression. miRAGDB is freely available and can be accessed at http://52.4.112.252/shiny/miragdb/.

The genome trilogy of Anopheles stephensi, an urban malaria vector, reveals structure of a locus associated with adaptation to environmental heterogeneity.

Anopheles stephensi is the most menacing malaria vector to watch for in newly urbanising parts of the world. Its fitness is reported to be a direct consequence of the vector adapting to laying eggs in over-head water tanks with street-side water puddles polluted by oil and sewage. Large frequent inversions in the genome of malaria vectors are implicated in adaptation. We report the genome assembly of a strain of An. stephensi of the type-form, collected from a construction site from Chennai (IndCh) in 2016. The genome reported here with a L50 of 4, completes the trilogy of high-resolution genomes of strains with respect to a 16.5 Mbp 2Rb genotype in An. stephensi known to be associated with adaptation to environmental heterogeneity. Unlike the reported genomes of two other strains, STE2 (2R+b/2Rb) and UCI (2Rb/2Rb), IndCh is found to be homozygous for the standard form (2R+b/2R+b). Comparative genome analysis revealed base-level details of the breakpoints and allowed extraction of 22,650 segregating SNPs for typing this inversion in populations. Whole genome sequencing of 82 individual mosquitoes from diverse geographical locations reveal that one third of both wild and laboratory populations maintain the heterozygous genotype of 2Rb. The large number of SNPs can be tailored to 1740 exonic SNPs enabling genotyping directly from transcriptome sequencing. The genome trilogy approach accelerated the study of fine structure and typing of an important inversion in An. stephensi, putting the genome resources for this understudied species on par with the extensively studied malaria vector, Anopheles gambiae. We argue that the IndCh genome is relevant for field translation work compared to those reported earlier by showing that individuals from diverse geographical locations cluster with IndCh, pointing to significant convergence resulting from travel and commerce between cities, perhaps, contributing to the survival of the fittest strain.

Hidden genomic features of an invasive malaria vector, Anopheles stephensi, revealed by a chromosome-level genome assembly.

BACKGROUND: The mosquito Anopheles stephensi is a vector of urban malaria in Asia that recently invaded Africa. Studying the genetic basis of vectorial capacity and engineering genetic interventions are both impeded by limitations of a vector's genome assembly. The existing assemblies of An. stephensi are draft-quality and contain thousands of sequence gaps, potentially missing genetic elements important for its biology and evolution. RESULTS: To access previously intractable genomic regions, we generated a reference-grade genome assembly and full transcript annotations that achieve a new standard for reference genomes of disease vectors. Here, we report novel species-specific transposable element (TE) families and insertions in functional genetic elements, demonstrating the widespread role of TEs in genome evolution and phenotypic variation. We discovered 29 previously hidden members of insecticide resistance genes, uncovering new candidate genetic elements for the widespread insecticide resistance observed in An. stephensi. We identified 2.4 Mb of the Y chromosome and seven new male-linked gene candidates, representing the most extensive coverage of the Y chromosome in any mosquito. By tracking full-length mRNA for > 15 days following blood feeding, we discover distinct roles of previously uncharacterized genes in blood metabolism and female reproduction. The Y-linked heterochromatin landscape reveals extensive accumulation of long-terminal repeat retrotransposons throughout the evolution and degeneration of this chromosome. Finally, we identify a novel Y-linked putative transcription factor that is expressed constitutively throughout male development and adulthood, suggesting an important role. CONCLUSION: Collectively, these results and resources underscore the significance of previously hidden genomic elements in the biology of malaria mosquitoes and will accelerate the development of genetic control strategies of malaria transmission.

A Near-Chromosome Level Genome Assembly of Anopheles stephensi.

Malaria remains a major healthcare risk to growing economies like India, and a chromosome-level reference genome of Anopheles stephensi is critical for successful vector management and understanding of vector evolution using comparative genomics. We report chromosome-level assemblies of an Indian strain, STE2, and a Pakistani strain SDA-500 by combining draft genomes of the two strains using a homology-based iterative approach. The resulting assembly IndV3/PakV3 with L50 of 9/12 and N50 6.3/6.9 Mb had scaffolds long enough for building 90% of the euchromatic regions of the three chromosomes, IndV3s/PakV3s, using low-resolution physical markers and enabled the generation of the next version of genome assemblies, IndV4/PakV4, using HiC data. We have validated these assemblies using contact maps against publicly available HiC raw data from two strains including STE2 and another lab strain of An. stephensi from UCI and compare the quality of the assemblies with other assemblies made available as preprints since the submission of the manuscript. We show that the IndV3s and IndV4 assemblies are sensitive in identifying a homozygous 2Rb inversion in the UCI strain and a 2Rb polymorphism in the STE2 strain. Multiple tandem copies of CYP6a14, 4c1, and 4c21 genes, implicated in insecticide resistance, lie within this inversion locus. Comparison of assembled genomes suggests a variation of 1 in 81 positions between the UCI and STE2 lab strains, 1 in 82 between SDA-500 and UCI strain, and 1 in 113 between SDA-500 and STE2 strains of An. stephensi, which are closer than 1 in 68 variations among individuals from two other lab strains sequenced and reported here. Based on the developmental transcriptome and orthology of all the 54 olfactory receptors (ORs) to those of other Anopheles species, we identify an OR with the potential for host recognition in the genus Anopheles. A comparative analysis of An. stephensi genomes with the completed genomes of a few other Anopheles species suggests limited inter-chromosomal gene flow and loss of synteny within chromosomal arms even among the closely related species.

Classification of Grain Amaranths Using Chromosome-Level Genome Assembly of Ramdana, A. hypochondriacus.

In the age of genomics-based crop improvement, a high-quality genome of a local landrace adapted to the local environmental conditions is critically important. Grain amaranths produce highly nutritional grains with a multitude of desirable properties including C4 photosynthesis highly sought-after in other crops. For improving the agronomic traits of grain amaranth and for the transfer of desirable traits to dicot crops, a reference genome of a local landrace is necessary. Toward this end, our lab had initiated sequencing the genome of Amaranthus (A.) hypochondriacus (A.hyp_K_white) and had reported a draft genome in 2014. We selected this landrace because it is well adapted for cultivation in India during the last century and is currently a candidate for TILLING-based crop improvement. More recently, a high-quality chromosome-level assembly of A. hypochondriacus (PI558499, Plainsman) was reported. Here, we report a chromosome-level assembly of A.hyp_K_white (AhKP) using low-coverage PacBio reads, contigs from the reported draft genome of A.hyp_K_white, raw HiC data and reference genome of Plainsman (A.hyp.V.2.1). The placement of A.hyp_K_white on the phylogenetic tree of grain amaranths of known accessions clearly suggests that A.hyp_K_white is genetically distal from Plainsman and is most closely related to the accession PI619259 from Nepal (Ramdana). Furthermore, the classification of another accession, Suvarna, adapted to the local environment and selected for yield and other desirable traits, is clearly Amaranthus cruentus. A classification based on hundreds of thousands of SNPs validated taxonomy-based classification for a majority of the accessions providing the opportunity for reclassification of a few.