title: Direct squencing from the minimal number of DNA molecules needed to fill a 454 picotiterplate.
abstract: The large amount of DNA needed to prepare a library in next generation sequencing protocols hinders direct sequencing of small DNA samples. This limitation is usually overcome by the enrichment of such samples with whole genome amplification (WGA), mostly by multiple displacement amplification (MDA) based on φ29 polymerase. However, this technique can be biased by the GC content of the sample and is prone to the development of chimeras as well as contamination during enrichment, which contributes to undesired noise during sequence data analysis, and also hampers the proper functional and/or taxonomic assignments. An alternative to MDA is direct DNA sequencing (DS), which represents the theoretical gold standard in genome sequencing. In this work, we explore the possibility of sequencing the genome of Escherichia coli fs 24 from the minimum number of DNA molecules required for pyrosequencing, according to the notion of one-bead-one-molecule. Using an optimized protocol for DS, we constructed a shotgun library containing the minimum number of DNA molecules needed to fill a selected region of a picotiterplate. We gathered most of the reference genome extension with uniform coverage. We compared the DS method with MDA applied to the same amount of starting DNA. As expected, MDA yielded a sparse and biased read distribution, with a very high amount of unassigned and unspecific DNA amplifications. The optimized DS protocol allows unbiased sequencing to be performed from samples with a very small amount of DNA.
authors.affiliation: Área de Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública), Valencia, Spain; Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Valencia, Spain; CIBER en Epidemiología y Salud Pública (CIBEResp), Madrid, Spain.
authors.forename: Mária
authors.initials: M
authors.lastname: Džunková
authors.affiliation: Área de Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública), Valencia, Spain; Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Valencia, Spain; Institut de Biologia Evolutiva, CSIC-Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Marc
authors.initials: M
authors.lastname: Garcia-Garcerà
authors.affiliation: Área de Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública), Valencia, Spain; Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Valencia, Spain.
authors.forename: Llúcia
authors.initials: L
authors.lastname: Martínez-Priego
authors.affiliation: Área de Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública), Valencia, Spain; Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Valencia, Spain; CIBER en Epidemiología y Salud Pública (CIBEResp), Madrid, Spain.
authors.forename: Giussepe
authors.initials: G
authors.lastname: D'Auria
authors.affiliation: Institut de Biologia Evolutiva, CSIC-Universitat Pompeu Fabra, Barcelona, Spain.
authors.forename: Francesc
authors.initials: F
authors.lastname: Calafell
authors.affiliation: Área de Genómica y Salud, Fundación para el Fomento de la Investigación Sanitaria y Biomédica de la Comunidad Valenciana (FISABIO-Salud Pública), Valencia, Spain; Instituto Cavanilles de Biodiversidad y Biología Evolutiva, Universitat de València, Valencia, Spain; CIBER en Epidemiología y Salud Pública (CIBEResp), Madrid, Spain.
authors.forename: Andrés
authors.initials: A
authors.lastname: Moya
mesh_terms: D002874:Chromosome Mapping; D016000:Cluster Analysis; D004926:Escherichia coli / Q000235:genetics; D015723:Gene Library; D016680:Genome, Bacterial / Q000235:genetics; D059014:High-Throughput Nucleotide Sequencing / Q000295:instrumentation* / Q000379:methods*; D017422:Sequence Analysis, DNA