Location: Crop Germplasm ResearchTitle: BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in Upland cotton Author
Submitted to: PLoS One
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 8/29/2013
Publication Date: 10/8/2013
Citation: Buyyarapu, R., Kantety, R.V., Yu, J., Xu, Z., Kohel, R.J., Percy, R.G., Macmil, S., Wiley, G.B., Roe, B.A., Sharma, G.C. 2013. BAC-pool sequencing and analysis of large segments of A12 and D12 homoeologous chromosomes in Upland cotton. PLoS One. 8(10): e76757. doi:10.1371/journal.pone.0076757. Interpretive Summary: Technologies to sequence DNA molecules in the nuclear genomes of plant species evolve rapidly, but no cost-effective sequencing strategy to produce accurate data has been tested on complex Gossypium genomes. This report is the first study to design a Sanger/454 hybrid approach that was applied to allotetraploid Upland cotton. One large genomic segment, consisting of a number of large-insert DNA clones (BAC), from each of a pair of homoeologous chromosomes (A12 and D12) of Upland cotton was developed and sequenced via Sanger, 454, and Sanger/454 hybrid methods. Detailed sequence analyses among the datasets indicate that Sanger/454 pool-wise hybrid assemblies provided genome researchers with better coverage of the sequenced regions as well as more comprehensive coverage of the results compared to the two individual methods alone. Pooled BAC sequencing using Sanger/454 hybrid strategy would offer new opportunity for effective sequencing and assembling of large complex genomes such as Upland cotton.
Technical Abstract: New and emerging next generation sequencing technologies have reduced sequencing costs, but there is room for additional approaches that can be applied to complex polyploid plant genomes. Large (about 2.5GB) and highly repetitive tetraploid genome of G. hirsutum is still cost-intensive with traditional BAC-by-BAC sequencing. Here we report on a hybrid BAC-pool sequencing effort based on large genomic segments (Ctg) sequencing using Sanger/454 hybrid approach, and we evaluated the efficiency of this strategy. Two BAC contigs, Ctg-465 (chromosome 26 or D12) and Ctg-3301 (chromosome 12 or A12) from the homoeologous cotton chromosomes were selected for sequencing. Three to four BAC clones were pooled and sequenced using the GS20 FLX instrument. Ctg-465 was sequenced with coverage approaching up to 'about 35X' coverage from 12 BAC clones with 4 clones per each pool. Similarly, Ctg-3301 sequence coverage was sequenced up to 'about 34.5X' coverage from 7 BAC clones with 4 and 3 clones under in each pool. Using Sanger's sequencing method, 17 BACs from both contigs were sequenced individually at 'about 2.8-3X' coverage to enable generation of longer contig sequences in the assembly. BAC-end sequences (BES) and anchored markers for these chromosomes were initially evaluated using the de novo assemblies created by Sanger, 454 and Sanger/454 hybrid methods. Sanger/454 pool-wise hybrid assemblies offered better coverage of the sequenced regions and provided comprehensive coverage of the results during this analysis compared to the two individual methods alone. Pooled BAC sequencing using Sanger/454 hybrid sequencing method provides an opportunity to reduce cost and time needed for de novo sequencing and assembling of large genomic segments.