Parallel Sequencing of Wolbachia wCer2 from Donor and Novel Hosts Reveals Multiple Incompatibility Factors and Genome Stability after Host Transfers.

The application of Wolbachia in insect pest and vector control requires the establishment of genotypically stable host associations. The cytoplasmic incompatibility (CI) inducing Wolbachia strain wCer2 naturally occurs in the cherry fruit fly Rhagoletis cerasi as co-infection with other strains and was transferred to other fruit fly species by embryonic microinjections. We obtained wCer2 genome data from its native and three novel hosts, Drosophila simulans, Drosophila melanogaster, and Ceratitis capitata and assessed its genome stability, characteristics, and CI factor (cif) genes. De novo assembly was successful from Wolbachia cell-enriched singly infected D. simulans embryos, with minimal host and other bacterial genome traces. The low yield of Wolbachia sequence reads from total genomic extracts of one multiply infected R. cerasi pupa and one singly infected C. capitata adult limited de novo assemblies but was sufficient for comparative analyses. Across hosts wCer2 was stable in genome synteny and content. Polymorphic nucleotide sites were found in wCer2 of each host; however, only one nucleotide was different between R. cerasi and C. capitata, and none between replicated D. simulans lines. The wCer2 genome is highly similar to wAu (D. simulans), wMel (D. melanogaster), and wRec (Drosophila recens). In contrast to wMel and wRec (each with one cif gene pair) and wAu (without any cif genes), wCer2 has three pairs of Type I cif genes, and one Type V cifB gene without a cifA complement. This may explain previously reported CI patterns of wCer2, including incomplete rescue of its own CI modification in three novel host species.

Scarcity of Recurrent Regulatory Driver Mutations in Colorectal Cancer Revealed by Targeted Deep Sequencing.

BACKGROUND: Genetic testing of cancer samples primarily focuses on protein-coding regions, despite most mutations arising in noncoding DNA. Noncoding mutations can be pathogenic if they disrupt gene regulation, but the benefits of assessing promoter mutations in driver genes by panel testing has not yet been established. This is especially the case in colorectal cancer, for which few putative driver variants at regulatory elements have been reported. METHODS: We designed a unique target capture sequencing panel of 39 colorectal cancer driver genes and their promoters, together with more than 35 megabases of regulatory elements focusing on gene promoters. Using this panel, we sequenced 95 colorectal cancer and matched normal samples at high depth, averaging 170× and 82× coverage, respectively. RESULTS: Our target capture sequencing design enabled improved coverage and variant detection across captured regions. We found cases with hereditary defects in mismatch and base excision repair due to deleterious germline coding variants, and we identified mutational spectra consistent with these repair deficiencies. Focusing on gene promoters and other regulatory regions, we found little evidence for base or region-specific recurrence of functional somatic mutations. Promoter elements, including TERT, harbored few mutations, with none showing strong functional evidence. Recurrent regulatory mutations were rare in our sequenced regions in colorectal cancer, though we highlight some candidate mutations for future functional studies. CONCLUSIONS: Our study supports recent findings that regulatory driver mutations are rare in many cancer types and suggests that the inclusion of promoter regions into cancer panel testing is currently likely to have limited clinical utility in colorectal cancer.

High-throughput Quant-iT PicoGreen assay using an automated liquid handling system.

Workflows in NGS facilities require high-standard practices and high-throughput pipelines to process the large number of samples received in a timely manner. Downstream protocols such as NGS library preparation require accurate estimation of nucleic acid concentrations, which can be achieved using fluorescent dye-based nucleic acid measurement. Here, we report a protocol for preparing a 384-well Quant-iT PicoGreen assay. The protocol allows the concentrations of 184 DNA samples to be measured simultaneously in duplicate in only 1 h using an Eppendorf epMotion 5075 liquid handling system. The advantages of this high-throughput approach include a reduction in both reagents (10x less reagents compared to a standard protocol) and time (3 h for 384 samples compared with 3 days).

Field study reveals core plant microbiota and relative importance of their drivers.

Harnessing plant microbiota can assist in sustainably increasing primary productivity to meet growing global demands for food and biofuel. However, development of rational microbiome-based approaches for improving crop yield and productivity is currently hindered by a lack of understanding of the major biotic and abiotic factors shaping the crop microbiome under relevant field conditions. We examined bacterial and fungal communities associated with both aerial (leaves, stalks) and belowground (roots, soil) compartments of four commercial sugarcane varieties (Saccharum spp.) grown in several growing regions in Australia. We identified drivers of the sugarcane microbiome under field conditions and evaluated whether the plants shared a core microbiome. Sugarcane-associated microbial assemblages were primarily determined by plant compartment, followed by growing region, crop age, variety and Yellow Canopy Syndrome (YCS). We detected a core set of microbiota and identified members of the core microbiome that were influenced by YCS incidence. Our study revealed key hub microorganisms in the core microbiome networks of sugarcane leaves, stalks, roots and rhizosphere soil despite location and time-associated shifts in the community assemblages. Elucidating their functional roles and identification of the keystone core microbiota that sustain plant health could provide a technological breakthrough for a sustainable increase in crop productivity.

RNA-Sequencing to Elucidate Early Patterns of Dysregulation Underlying the Onset of Alzheimer's Disease.

With its ability to perform rapid transcriptome profiling and profound transcriptomic analysis powered by high-throughput sequencing at a high resolution with deep coverage, the advent of RNA sequencing technology, RNA-Seq, outperforms other methods in the field, such as microarrays, and has changed our way of performing transcriptomic investigation. Protocols for preparing libraries for RNA-Seq using the Illumina and Roche 454 sequencing platforms are included in this chapter. Common steps for library preparation in both platforms include RNA fragmentation, cDNA synthesis, adaptor ligation, and PCR amplification of cDNA strands. Illumina adopts solid-phase bridge PCR amplification, while 454 uses water-in-oil emulsion-based PCR amplification. Despite differences in the PCR amplification step, both platforms employ the same sequencing-by-synthesis technology for the sequencing process. Application of the RNA-Seq technique in the context of dysregulation of the transcriptome in Alzheimer's disease is also discussed.

Sequencing of hippocampal and cerebellar transcriptomes provides new insights into the complexity of gene regulation in the human brain.

The hippocampus and cerebellum represent anatomically and functionally distinct parts of the human brain. The RNA-Seq technique makes it possible to investigate the human transcriptome with unprecedented resolution, allowing identification of differential mRNA splicing and promoter usage on a genome-wide scale. We undertook whole-mRNA sequencing of samples from the human hippocampus and cerebellum. A bioinformatic analysis revealed distinct expression patterns of genes related to the molecular physiology of neurons and glial cells. Upregulated genes in hippocampal tissue included serpin peptidase inhibitor, clade A (SERPINA3), lymphocyte antigen 6 complex, locus H (LY6H) and transthyretin (TTR). In cerebellum, the cerebellin 3 precursor (CLBN3) and Zic family member 4 (ZIC4) genes were significantly upregulated. These changes were validated in independent donor samples by qRT-PCR. The hippocampus and the cerebellum showed striking differences in splicing patterns and promoter usage. A notable example of this was the gene for NGFI-A binding protein 2 (NAB2), which displayed tissue-specific isoforms which may affect its function as a transcriptional repressor.

RNA-Seq analysis of the parietal cortex in Alzheimer's disease reveals alternatively spliced isoforms related to lipid metabolism.

The parietal cortex of the human brain plays a unique role in the coordination of movement and in the integration of signals from the other cortices. Because of its extensive connections and involvement in many higher-order cognitive functions, neurodegenerative changes in the parietal lobe are believed to be crucial in the early symptoms of Alzheimer's disease (AD). Little is known about the transcriptome of this part of the human brain or how it is perturbed by the neurodegenerative process. To that end, we performed mRNA sequencing using the Illumina RNA-Seq technique on samples derived from normal and AD parietal lobes. Gene expression analysis evaluating alternatively spliced isoform expression and promoter usage revealed surprisingly elevated transcriptome activity in the AD condition. This phenomenon was particularly apparent in the alternative usage of transcriptional start sites. A Gene Ontology analysis of the differentially expressed genes revealed enrichment in the functional pathways related to lipid metabolism, thus highlighting the importance of astrocyte activity in the neurodegenerative process. We also identified an upregulation of the diazepam-binding inhibitor (DBI) gene in AD, as the result of a splicing switch toward shorter, intron-retaining isoforms driven by alternative promoters and was coupled with a simultaneous decrease in the abundance of protein-coding transcripts. These two DBI isoforms have not been described previously.