Barcoding-free BAC Pooling Enables Combinatorial Selective Sequencing of the Barley Gene Space

We propose a new sequencing protocol that combines recent advances in combinatorial pooling design and second-generation sequencing technology to efficiently approach de novo selective genome sequencing. We show that combinatorial pooling is a cost-effective and practical alternative to exhaustive DNA barcoding when dealing with hundreds or thousands of DNA samples, such as genome-tiling generich BAC clones. The novelty of the protocol hinges on the computational ability to efficiently compare hundreds of million of short reads and assign them to the correct BAC clones 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 extremely accurate (99.57% of the deconvoluted reads are assigned to the correct BAC), and the resulting BAC assemblies have very high quality (BACs are covered by contigs over about 77% of their length, on average). Experimental results on real data for a gene-rich subset of the barley genome confirm that the deconvolution is accurate (almost 70% of left/right pairs in paired-end reads are assigned to the same BAC, despite being processed independently) and the BAC assemblies have good quality (the average sum of all assembled contigs is about 88% of the estimated BAC length). Data availability: Barley raw sequencing data for one set of 2,197 MTP gene-enriched BACs can be obtained from NCBI Sequence Read Archive (http://www.ncbi.nlm.nih.gov/sra?term=(SRA047913))