improving-small-rna-seq-less-bias-better-detection-2-o-methyl

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

The study of small RNAs (sRNAs) by next-generation sequencing (NGS) is challenged by bias issues during library preparation. Several types of sRNA such as ...

Université Paris-Saclay

In mammals, neuroepithelial cells play an essential role in embryonic neurogenesis, whereas glial stem cells are the principal source of neurons at postembryonic stages. By contrast, neuroepithelial-like stem/progenitor (NE) cells have been shown to be present throughout life in teleosts. We used three-dimensional (3D) reconstructions of cleared transgenic wdr12:GFP medaka brains to demonstrate that this cell type is widespread in juvenile and to identify new regions containing NE cells. We established the gene expression profile of optic tectum (OT) NE cells by cell sorting followed by RNA-seq. Our results demonstrate that most OT NE cells are indeed active stem cells and that some of them exhibit long G2 phases. We identified several novel pathways (e.g., DNA repair pathways) potentially involved in NE cell homeostasis. In situ hybridization studies showed that all NE populations in the postembryonic medaka brain have a similar molecular signature. Our findings highlight the importance of NE progenitors in medaka and improve our understanding of NE-cell biology. These cells are potentially useful not only for neural stem cell studies but also for improving the characterization of neurodevelopmental diseases, such as microcephaly. Stem Cells 2017;35:1505-1518.

Postembryonic Fish Brain Proliferation Zones Exhibit Neuroepithelial-Type Gene Expression Profile.

Next-generation sequencing technologies have revolutionized the study of small RNAs (sRNAs) on a genome-wide scale. However, classical sRNA library preparation methods introduce serious bias, mainly during adapter ligation steps. Several types of sRNA including plant microRNAs (miRNA), piwi-interacting RNAs (piRNA) in insects, nematodes and mammals, and small interfering RNAs (siRNA) in insects and plants contain a 2'-O-methyl (2'-OMe) modification at their 3' terminal nucleotide. This inhibits 3' adapter ligation and makes library preparation particularly challenging. To reduce bias, the NEBNext kit (New England Biolabs) uses polyethylene glycol (PEG), the NEXTflex V2 kit (BIOO Scientific) uses both randomised adapters and PEG, and the novel SMARTer (Clontech) and CATS (Diagenode) kits avoid ligation altogether. Here we compared these methods with Illumina's classical TruSeq protocol regarding the detection of normal and 2' OMe RNAs. In addition, we modified the TruSeq and NEXTflex protocols to identify conditions that improve performance.

Systematic comparison of small RNA library preparation protocols for next-generation sequencing.

The mitochondrial genomes of  strains contain up to 13 introns. An intronless recombinant genome introduced into the nuclear background of  strain W303 gave the  CW252 strain, which is used to model mitochondrial respiratory pathologies. The complete sequence of this mitochondrial genome was obtained using a hybrid assembling methodology.

Complete Sequence of the Intronless Mitochondrial Genome of the Saccharomyces cerevisiae Strain CW252.

Genome replication is highly regulated in time and space, but the rules governing the remodeling of these programs during evolution remain largely unknown. We generated genome-wide replication timing profiles for ten Lachancea yeasts, covering a continuous evolutionary range from closely related to more divergent species. We show that replication programs primarily evolve through a highly dynamic evolutionary renewal of the cohort of active replication origins. We found that gained origins appear with low activity yet become more efficient and fire earlier as they evolutionarily age. By contrast, origins that are lost comprise the complete range of firing strength. Additionally, they preferentially occur in close vicinity to strong origins. Interestingly, despite high evolutionary turnover, active replication origins remain regularly spaced along chromosomes in all species, suggesting that origin distribution is optimized to limit large inter-origin intervals. We propose a model on the evolutionary birth, death, and conservation of active replication origins.

The evolution of the temporal program of genome replication.

Forty years ago the advent of Sanger sequencing was revolutionary as it allowed complete genome sequences to be deciphered for the first time. A second revolution came when next-generation sequencing (NGS) technologies appeared, which made genome sequencing much cheaper and faster. However, NGS methods have several drawbacks and pitfalls, most notably their short reads. Recently, third-generation/long-read methods appeared, which can produce genome assemblies of unprecedented quality. Moreover, these technologies can directly detect epigenetic modifications on native DNA and allow whole-transcript sequencing without the need for assembly. This marks the third revolution in sequencing technology. Here we review and compare the various long-read methods. We discuss their applications and their respective strengths and weaknesses and provide future perspectives.

Claude Thermes

Institute for Integrative Biology of the Cell,
UMR9198,
CNRS CEA Univ Paris-Sud,
Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud

Université Paris-Saclay

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Claude Thermes

Institute for Integrative Biology of the Cell,UMR9198,CNRS CEA Univ Paris-Sud,Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud

Université Paris-Saclay

Improving Small RNA-seq: Less Bias and Better Detection of 2'-O-Methyl RNAs

Institute for Integrative Biology of the Cell,
UMR9198,
CNRS CEA Univ Paris-Sud,
Institute for Integrative Biology of the Cell, UMR9198, CNRS CEA Univ Paris-Sud