2019 II CIMUS workshop: Starting up a long sequencing facility

Abstract: After a decade of absolute domain of short read NGS sequencing, there are still unsolved questions about the structure and organization of the DNA that the new generation of long read sequencers are helping to address. Perhaps one of the most astonishing technologies, both by its concept and accessibility is Oxford Nanopore Technologies (ONT) sequencing. This platform is based on disposable flowcells constituted by an artificial bilipidic membrane containing multiple nano-scale pores. These pores allow the flow of either DNA or RNA while registering the subsequent ionic current. The measurement of the subtle changes in this ionic current allows deciphering the molecule sequence at nucleotide level, generating long-read data. In this work, we explain process of establishing an ONT MinION at CIMUS. Since the foundation of this facility, we've overcomed four big challenges: The constant evolution and improvement of this technology affecting both chemistry and bioinfomatic pipelines, achieving a suitable DNA isolation protocol for long-read sequencing, improving the already per se delicate library construction and dealing with bottlenecks on processing and storing the massive amount of data generated in each run. To this day our group has undertaken the sequencing of more than a hundred DNA samples from a miriad of species, such as healthy and tumor human tissues and cell lines, dog, bivalve, yeast, Escherichia coli or lambda phage. Additionally, this technology has proven to be extremely versatile for the massive sequencing of high molecular weight PCR barcoded fragments. Among our greatest achievements, this technology has allowed us the de-novo assembly of a non model animal (Cerastoderma edule), the validation of retraspositions insertions and movilizations in cancer, the study of structural variations, otherwise unachievable with short reads, and the massive and simultaneous sequencing of hundreds of mitochondrial DNA amplicons to establish molecular phylogenies. Nowdays, we rutinary obtain from 10 to 25 Gbases per each run, with average fragment lengts up to 20 K bases and some reads up to 400 Kbases.

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