ORC then recruits Cell Division Cycle 6 (Cdc6), chromatin licensing and DNA replication factor 1 (Cdt1), and the replication helicase subunits Mcm2–7 to form a pre-replicative complex (pre-RC) 5. In eukaryotes, the six subunit ORC complex (comprising Orc1–6) assembles on DNA prior to S-phase 4. Nevertheless, the proteins involved in the initiation of DNA replication are highly conserved. By contrast, replication origins are sequence non-specific in plants and animals, in the fission yeast Schizosaccharomyces pombe, and even in many other budding yeasts and fungi 3. Such sequences enable the replication of extrachromosomal plasmids and are thus termed autonomously replicating sequences (ARSs) 2. In the budding yeast Saccharomyces cerevisiae, replication origins are specified by DNA sequence motifs that comprise an essential A element (about 11nt in length) and multiple B elements 1. The DNA sequence specificity of replication origins, mediated by the Orc4 α-helix, has co-evolved with the gain of ORC-Sir4-mediated gene silencing and the loss of RNA interference. cerevisiae and Orc4 α-helix mutations change genome-wide origin firing patterns. Using a massively parallel origin selection assay coupled with a custom mutual-information-based modeling approach, and a separate analysis of whole-genome replication profiling, here we show that the Orc4 α-helix contributes to the DNA sequence-specificity of origins in S. cerevisiae ORC-Cdc6-Cdt1-Mcm2-7 (OCCM) bound to origin DNA revealed that a loop within Orc2 inserts into a DNA minor groove and an α-helix within Orc4 inserts into a DNA major groove. Origin establishment requires well-defined DNA sequence motifs in Saccharomyces cerevisiae and some other budding yeasts, but most eukaryotes lack sequence-specific origins. All rights reserved.DNA replication in eukaryotic cells initiates from replication origins that bind the Origin Recognition Complex (ORC). ![]() This validation study establishes that ONT MinION sequence data used in conjunction with the NGSpeciesID pipeline can produce consensus DNA sequences of sufficient accuracy for forensic genetic species identification.īioinformatic pipeline DNA barcoding High-throughput sequencing (HTS) MinION MtDNA NGSpeciesID Species identification Validation.Ĭopyright © 2021 The Authors. Finally, we investigated the impact of differences between the MinION consensus and Sanger control sequences on correct species identification to understand the ability and accuracy of the MinION consensus sequence to differentiate the true species from the next most similar species. Second, we systematically evaluated the read variation distribution around the generated consensus sequences to understand what confidence we have in the accuracy of the resulting consensus sequence and to determine how to interpret individual sample results. First, we tested whether the clustering-based bioinformatics pipeline NGSpeciesID can be used to generate an accurate consensus sequence for species identification. Here, we present a MinION deep read sequence data validation study for species identification. However, there has been no formal validation of forensic species identification using high-throughput (deep read) sequence data from the MinION making it currently impractical for many wildlife forensic end-users. The Oxford Nanopore Technologies (ONT) MinION™ is an affordable and small footprint DNA sequencing device with the potential to quickly deliver reliable and cost effective data. However, the generation of HTS data to date requires expensive equipment and is cost-effective only when large numbers of samples are analysed simultaneously. HTS sequencing, which now dominates molecular biology research, has already been demonstrated for use in a number of forensic genetic analysis applications, including species identification. The gold standard for forensic genetics is conventional Sanger sequencing however, this is gradually being replaced by high-throughput sequencing (HTS) approaches which can generate millions of individual reads in a single experiment. ![]() Species identification of non-human biological evidence through DNA nucleotide sequencing is routinely used for forensic genetic analysis to support law enforcement.
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