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Combinatorial Pooling Enables Selective Sequencing of the Barley Gene Space
- Source :
- PLoS Computational Biology, Vol 9, Iss 4, p e1003010 (2013), PLoS computational biology, vol 9, iss 4, PLoS Computational Biology
- Publication Year :
- 2013
- Publisher :
- Public Library of Science (PLoS), 2013.
-
Abstract
- For the vast majority of species – including many economically or ecologically important organisms, progress in biological research is hampered due to the lack of a reference genome sequence. Despite recent advances in sequencing technologies, several factors still limit the availability of such a critical resource. At the same time, many research groups and international consortia have already produced BAC libraries and physical maps and now are in a position to proceed with the development of whole-genome sequences organized around a physical map anchored to a genetic map. We propose a BAC-by-BAC sequencing protocol that combines combinatorial pooling design and second-generation sequencing technology to efficiently approach denovo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when preparing sequencing libraries for hundreds or thousands of DNA samples, such as in this case gene-bearing minimum-tiling-path BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundred millions of short reads and assign them to the correct BAC clones (deconvolution) so that the assembly can be carried out clone-by-clone. Experimental results on simulated data for the rice genome show that the deconvolution is very accurate, and the resulting BAC assemblies have high quality. Results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate and the BAC assemblies have good quality. While our method cannot provide the level of completeness that one would achieve with a comprehensive whole-genome sequencing project, we show that it is quite successful in reconstructing the gene sequences within BACs. In the case of plants such as barley, this level of sequence knowledge is sufficient to support critical end-point objectives such as map-based cloning and marker-assisted breeding.<br />Author Summary The problem of obtaining the full genomic sequence of an organism has been solved either via a global brute-force approach (called whole-genome shotgun) or by a divide-and-conquer strategy (called clone-by-clone). Both approaches have advantages and disadvantages in terms of cost, manual labor, and the ability to deal with sequencing errors and highly repetitive regions of the genome. With the advent of second-generation sequencing instruments, the whole-genome shotgun approach has been the preferred choice. The clone-by-clone strategy is, however, still very relevant for large complex genomes. In fact, several research groups and international consortia have produced clone libraries and physical maps for many economically or ecologically important organisms and now are in a position to proceed with sequencing. In this manuscript, we demonstrate the feasibility of this approach on the gene-space of a large, very repetitive plant genome. The novelty of our approach is that, in order to take advantage of the throughput of the current generation of sequencing instruments, we pool hundreds of clones using a special type of “smart” pooling design that allows one to establish with high accuracy the source clone from the sequenced reads in a pool. Extensive simulations and experimental results support our claims.
- Subjects :
- 0106 biological sciences
Chromosomes, Artificial, Bacterial
Pooling
01 natural sciences
Genome
Mathematical Sciences
Contig Mapping
Models
Genomic library
Genome Sequencing
Cloning, Molecular
lcsh:QH301-705.5
Genetics
Genomic Library
0303 health sciences
Ecology
Bacterial
food and beverages
DNA sequencing theory
Genomics
Biological Sciences
Physical Chromosome Mapping
Computational Theory and Mathematics
Modeling and Simulation
Artificial
Sequence Analysis
Algorithms
Research Article
Biotechnology
Genetic Markers
Bioinformatics
Sequence analysis
Oryza sativa
Hybrid genome assembly
Computational biology
Biology
Genes, Plant
Chromosomes
DNA sequencing
03 medical and health sciences
Cellular and Molecular Neuroscience
Species Specificity
Genetic
Information and Computing Sciences
Computer Simulation
Molecular Biology
Ecology, Evolution, Behavior and Systematics
030304 developmental biology
Models, Genetic
Discrete Mathematics
Human Genome
Molecular
Computational Biology
Hordeum
Oryza
Sequence Analysis, DNA
DNA
Plant
Genes
lcsh:Biology (General)
Combinatorics
Computer Science
Generic health relevance
Mathematics
Cloning
010606 plant biology & botany
Subjects
Details
- ISSN :
- 15537358
- Volume :
- 9
- Database :
- OpenAIRE
- Journal :
- PLoS Computational Biology
- Accession number :
- edsair.doi.dedup.....7ad076df6082048abd225035594c479d