Biology subjects

Bayega, A.

Publications and source records attributed to Bayega, A..

5 recordsLinked to original sources

bioRxiv2025

Long-read RNA-seq delineates temporal transcriptional dynamics in multiplexed and sexed single medfly embryos

Long-read RNA sequencing has great potential to improve genomic characterization of non-model organisms due to its ability to yield full-length genes. Coupled with absolute gene expression quantification, dynamics of development orchestrated at transcript level can be elucidated with high precision. The resolution of this precision can be further improved by studying organisms as close as possible to their basic entities, single cells for example or single embryos. Here, we collected developing embryos of the Mediterranean fruit fly (medfly, Ceratitis capitata) at hourly time-points for the first 15 hours of development. The medfly is an organism of huge economic importance in agriculture due to its wide host range including apples, pear, citrus, olives, etc. We simultaneously isolated total RNA and genomic DNA from single embryos and sexed the embryos using Y-specific PCR assays. The RNA, spiked with external ERCC standards to aid in absolute quantification, was used to perform Nanopore long-read RNA-seq. We developed a genome-guided transcriptome assembly based on full-length transcripts and identified a total of 22,875 transcripts comprising 3879 novel genes, missed in the current NCBI predicted gene models. We show that, indeed, the absolute quantification of gene expression performs superiorly to relative quantification in highly dynamic systems such as developing embryos. Further, we used unsupervised clustering and lineage tracing algorithms to group and accurately place embryos along a pseudo-temporal development trajectory. We show that medfly embryos undergo successive waves of zygotic genome activation. We discover a dramatic reorganization of maternally deposited mRNA occurring within the first 3 hours of egg laying followed by maternal-to-zygotic transition. We finally identify modules of temporal synexpression and elucidate the biological role of these modules. Together, these results provide the first detailed look at early embryo development in the medfly and should aid in future control efforts of this pest.

genomics↗

bioRxiv2025

Identification of Novel DNA Sequence Motifs that Modulate Transcription in T cells

Considerable progress has been made towards associating transcription factor binding sites (TFBS) with cell-type-specific gene expression, however, the full repertoire of DNA sequence motifs that regulate transcription remains unknown. Improving our understanding of transcriptional regulation is especially important in T cells, given the enormous potential of genetically engineered T cells as an emerging class of therapeutics. Here, we report results from a comprehensive and unbiased survey investigating whether there are novel motifs enriched in regulatory regions of genes with the highest constitutive and selective expression across diverse T- cell subsets. Using computational and experimental methods, we identified 2,036 novel motifs and 629 previously curated TFBS that are enriched, both individually and in specific combinations, in the regulatory regions of genes exhibiting T-cell-specific gene expression. We then used the self-transcribing active regulatory region sequencing (STARR-seq) assay to evaluate all possible three- way combinations of a subset of 18 candidate motifs to test their ability to modulate transcription in immortalized lymphoblastic cell lines of T-cell origin (Jurkat E6) versus myeloid origin (K562). Our results revealed novel motifs that modulate gene transcription in T cells, with some exhibiting stronger regulatory effects than TFBS for TFs with established roles in T cells. The regulatory activity of these novel motifs was influenced by the motifs orientation, position, and copy number. Overall, these results highlight our incomplete understanding of the relationship between sequence composition and T-cell gene regulation and indicate that previously annotated TFBS represent only a subset of motifs capable of modulating gene transcription in T cells.

bioinformatics↗

bioRxiv2022

Improved Nanopore full-length cDNA sequencing by PCR-suppression

Full-length transcript sequencing remains a main goal of RNA sequencing. However, even the application of long-read sequencing technologies such as Oxford Nanopore Technologies still fail to yield full-length transcript sequencing for a significant portion of sequenced reads. Since these technologies can sequence reads that are far longer than the longest known processed transcripts, the lack of efficiency to obtain full-length transcripts from good quality RNAs stems from library preparation inefficiency rather than the presence of degraded RNA molecules. It has previously been shown that addition of inverted terminal repeats in cDNA during reverse transcription followed by single-primer PCR creates a PCR suppression effect that prevents amplification of short molecules thus enriching the library for longer transcripts. We adapted this method for Nanopore cDNA library preparation and show that not only is PCR efficiency increased but gene body coverage is dramatically improved. The results show that implementation of this simple strategy will result in better quality full-length RNA sequencing data and make full-length transcript sequencing possible for most of sequenced reads. Contribution to the fieldLong-read RNA sequencing aims to sequence expressed transcripts in their entirety. However, this has remained a challenge, mainly due to inherent inefficiencies in cDNA library preparation. Herein, we provide a new Nanopore cDNA library preparation protocol, termed Panhandle, that improves the efficiency of cDNA PCR with yields 2 - 8 times the yields obtained with ordinary PCR. This is key, as this should in turn reflect in the possibility of lowering the number of PCR cycles needed to obtain ample sequencing material, which in turn could reduce PCR biases, PCR artifacts, turnaround time, reagents, and could increase general quality of the library. Further, transcripts generated using the Panhandle method show better gene body coverage and more accurate transcription start site mapping than regular methods. This represents an important step towards full-length cDNA sequencing by Nanopore.

genomics↗

bioRxiv2018

De novo genome assembly of the olive fruit fly (Bactrocera oleae) developed through a combination of linked-reads and long-read technologies

Long-read sequencing has greatly contributed to the generation of high quality assemblies, albeit at a high cost. It is also not always clear how to combine sequencing platforms. We sequenced the genome of the olive fruit fly (Bactrocera oleae), the most important pest in the olive fruits agribusiness industry, using Illumina short-reads, mate-pairs, 10x Genomics linked-reads, Pacific Biosciences (PacBio), and Oxford Nanopore Technologies (ONT). The 10x linked-reads assembly gave the most contiguous assembly with an N50 of 2.16 Mb. Scaffolding the linked-reads assembly using long-reads from ONT gave a more contiguous assembly with scaffold N50 of 4.59 Mb. We also present the most extensive transcriptome datasets of the olive fly derived from different tissues and stages of development. Finally, we used the Chromosome Quotient method to identify Y-chromosome scaffolds and show that the long-reads based assembly generates very highly contiguous Y-chromosome assembly.\n\nJR is a member of the MinION Access Program (MAP) and has received free-of-charge flow cells and sequencing kits from Oxford Nanopore Technologies for other projects. JR has had no other financial support from ONT.\n\nAB has received re-imbursement for travel costs associated with attending Nanopore Community meeting 2018, a meeting organized my Oxford Nanopore Technologies.

genomics↗

bioRxiv2018

Transcriptome landscape of the developing olive fruit fly embryo delineated by Oxford Nanopore long-read RNA-Seq

The olive fruit fly or olive fly (Bactrocera oleae) is the most important pest of cultivated olive trees. Like all insects the olive fly undergoes complete metamorphosis. However, the transcription dynamics that occur during early embryonic development have not been explored, while detailed transcriptomic analysis in the absence of a fully annotated genome is challenging. We collected olive fly embryos at hourly intervals for the first 6 hours of development and performed full-length cDNA-Seq using a purpose designed SMARTer cDNA synthesis protocol followed by sequencing on the MinION (Oxford Nanopore Technologies). We generated 31 million total reads across the timepoints (median yield 4.2 million per timepoint). The reads showed 98 % alignment rate to the olive fly genome and 91 % alignment rate to the NBCI predicted B. oleae gene models. Over 50 % of the expressed genes had at least one read covering its entire length validating our full-length RNA-Seq procedure. Expression of 68 % of the predicted B. oleae genes was detected in the first six hours of development. We generated a de novo transcriptome assembly of the olive fly and identified 3553 novel genes and a total of 79,810 transcripts; a fourfold increase in transcriptome diversity compared to the NCBI predicted transcriptome. On a global scale, the first six hours of embryo development were characterized by dramatic transcriptome changes with the total number of transcripts per embryo dropping to half from the first hour to the second hour of embryo development. Clustering of genes based on temporal co-expression followed by gene-set enrichment analysiss of genes expressed in the first six hours of embryo development showed that genes involved in transcription and translation, macro-molecule biosynthesis, and neurodevelopment were highly enriched. These data provide the first insight into the transcriptome landscape of the developing olive fly embryo. The data also reveal transcript signatures of sex development. Overall, full-length sequencing of the cDNA molecules permitted a detailed characterization of the isoform complexity and the transcriptional dynamics of the first embryonic stages of the B. oleae.

developmental biology↗