TransFlow: a modular framework for assembling and assessing accurate de novo transcriptomes in non-model organisms

[Background]: The advances in high-throughput sequencing technologies are allowing more and more de novo assembling of transcriptomes from many new organisms. Some degree of automation and evaluation is required to warrant reproducibility, repetitivity and the selection of the best possible transcriptome. Workflows and pipelines are becoming an absolute requirement for such a purpose, but the issue of assembling evaluation for de novo transcriptomes in organisms lacking a sequenced genome remains unsolved. An automated, reproducible and flexible framework called TransFlow to accomplish this task is described.[Results]: TransFlow with its five independent modules was designed to build different workflows depending on the nature of the original reads. This architecture enables different combinations of Illumina and Roche/454 sequencing data, and can be extended to other sequencing platforms. Its capabilities are illustrated with the selection of reliable plant reference transcriptomes and the assembling six transcriptomes (three case studies for grapevine leaves, olive tree pollen, and chestnut stem, and other three for haustorium, epiphytic structures and their combination for the phytopathogenic fungus Podosphaera xanthii). Arabidopsis and poplar transcriptomes revealed to be the best references. A common result regarding de novo assemblies is that Illumina paired-end reads of 100 nt in length assembled with OASES can provide reliable transcriptomes, while the contribution of longer reads is noticeable only when they complement a set of short, single-reads.[Conclusions]:TransFlow can handle up to 181 different assembling strategies. Evaluation based on principal component analyses allows its self-adaptation to different sets of reads to provide a suitable transcriptome for each combination of reads and assemblers. As a result, each case study has its own behaviour, prioritises evaluation parameters, and gives an objective and automated way for detecting the best transcriptome within a pool of them. Sequencing data type and quantity (preferably several hundred millions of 2×100 nt or longer), assemblers (OASES for Illumina, MIRA4 and EULER-SR reconciled with CAP3 for Roche/454) and strategy (preferably scaffolding with OASES, and probably merging with Roche/454 when available) arise as the most impacting factors.This work was supported by co-funding by the European Union through the European Regional Development Fund (ERDF) 2014-2020 “Programa Operativo de Crecimiento Inteligente” together with Spanish AEI “Agencia Estatal de Investigación” (BFU2016-77243-P, RTC-2015-4181-2, RTC-2016-4824-2, AGL2013-41939-R and AGL2016-76216-C2-1-R), AEI-INIA (RTA2013-00023-C02 and RTA2013-00068-C03), and Junta de Andalucía (P2011-CVI-7487), as well as CSIC grant 201540E065. PS received a postdoctoral fellowship from Junta de Andalucía linked to grant P10-CVI-6075. Publication costs were funded by the mentioned RTA2013-00068-C03 grant

Anales de Edafología y Agrobiología Tomo 30 Número 3-4

Determinación conjunta de fósforo, potasio, calcio, magnesio, hierro, manganeso, cobre y cinc en plantas, por F. Duque Macías.-- Actividad microbiológica del humus en suelos semiáridos, por F. Velasco de Pedro y J. M. Lozano Calle.-- Estudio mineralógico de las formaciones superficiales del páramo calizo, por J. Pérez Mateos y J. Vaudour.-- Formas de nitrógeno en los suelos volcánicos de Sibundoy, vertiente andina del Amazonas colombiano, por M. Blasco L., F. Romo L., O. Bastidas O. y A. Caicedo V.-- Acerca de la Scorzonera graminofolia L. en relación con su contenido en caucho, por N. Velázquez Sánchez y M. R. Felipe Antón.-- Población mIcrobiana y tipos de humus en suelos semiúridos, por J. N. Lozano Calle y F. Velasco de Pedro.-- Medida comparada de los calores de inmersión y de adsorción del sistema benceno-geles de sílice, por G. Dios Cancela, F. Rouquerol y J. Rouquerol.--Suelos de la zona húmeda española. V l. Pelosol, por F. Guitián Ojea, T. Carballas y A. Pérez Pujalte.-- Análisis de las aguas de percolación a través del suelo en condiciones contraladas, por F. Guitián Ojea, F. Rodríguez Seoane y F. Díaz Fierros.-- Interestratificación de treS componentes en silicatos de estrutctura laminar. II, por A. Ruiz Amil, A.. Guerrero Laverat y D. M. C. Mac Ewan.-- Atapulgita y sepiolita en suelos sobre sedimentos calizos de Salamanca (España), por M. Sánchez Camazano y A. García Rodríguez.-- Relaciones fisiológicas en plantas pratenses y pascícolas de comunidades semina turales de la provincia de Salamanca, por B. García Criado, F. Duque Macías, y J. M. Gómez Gutiérrez.-- Notas. ¿Existen suelos mediterráneos en España?.-- Nuevos consejeros del Patronato Alonso de Herrera.-- Elecciones para renovación de la Junta de Gobierno del Patronato Alonso de Herrera.-- Próxima reunión del Pleno del Patronato Alonso de Herrera.-- Política científica del Patronato.-- Fallecimiento del consejero técnico Sr. Huarte.-- Donación de la Fundación a Alexander von Humboldt» al Centro de Edafología y Biología Aplicada de! Segura.-- Secuencias de la Reunión Hispano-Belga de minerales de la arcilla.-- Seminario sobre micromorfología del suelo.-- El Profesor Webb visita varios centros del Instituto Nacional de Edafología.-- Reunión del Comité Interinstitutos de Análisis Foliar.-- España exporta investigación.-- Creación de la Sección de Geoquímica y Génesis Mineral en el Instituto de Edafología de Madrid.-- Invitación a profesores extranjeros.-- Reuniones, Congresos y Becas.-- Viajes del Dr. Cadahía.-- Cobre radioactiva disponible en INCRA.-- Instituto de aclimatación de AlmeríaPeer reviewe

Accurate whole human genome sequencing using reversible terminator chemistry

DNA sequence information underpins genetic research, enabling discoveries of important biological or medical benefit. Sequencing projects have traditionally used long (400-800 base pair) reads, but the existence of reference sequences for the human and many other genomes makes it possible to develop new, fast approaches to re-sequencing, whereby shorter reads are compared to a reference to identify intraspecies genetic variation. Here we report an approach that generates several billion bases of accurate nucleotide sequence per experiment at low cost. Single molecules of DNA are attached to a flat surface, amplified in situ and used as templates for synthetic sequencing with fluorescent reversible terminator deoxyribonucleotides. Images of the surface are analysed to generate high-quality sequence. We demonstrate application of this approach to human genome sequencing on flow-sorted X chromosomes and then scale the approach to determine the genome sequence of a male Yoruba from Ibadan, Nigeria. We build an accurate consensus sequence from >30x average depth of paired 35-base reads. We characterize four million single-nucleotide polymorphisms and four hundred thousand structural variants, many of which were previously unknown. Our approach is effective for accurate, rapid and economical whole-genome re-sequencing and many other biomedical applications