Pacific Biosciences and KeyGene collaborate on tetraploid cotton genome assembly and transcriptome map

KeyGene and Pacific Biosciences recently announced completion of the integrated genome and transcriptome of tetraploid cotton (G. hirsutum). The results of this collaboration hallmark the first example of a high-quality genome assembly for a polyploid crop larger than 2 Gbp. This was achieved via an integrated genome and transcriptome sequencing approach using long read sequences in combination with DNA based physical maps. The genome assembly was generated with almost 90% of the sequenced genome assembling into only 1207 sequence contigs / BAC superscaffolds. This improved the cotton genome assembly with an impressive factor 842 when compared with conventional short read approaches. The results described here were presented at the Plant and Animal Genome (PAG) conference held in San Diego, USA on January 13, 2015.

The companies joined forces to unravel the complex genome of this important fiber crop, leveraging Pacific Biosciences’ long read PacBio® RS II sequencing platform for genome sequencing in combination with KeyGene’s Whole Genome Profiling (WGP™) method that generates high quality DNA-based physical maps based on partially sequenced BAC fragments. Assembly of long read PacBio RS II sequences (generated by 38x genome coverage sequencing) yielded a more than 47-fold reduction of the number of sequence contigs when compared with conventional short read / paired end sequence assemblies based on 293x genome coverage. Integration of the long read sequence contigs with the WGP physical map (based on BACs representing a 10x genome coverage) resulted in a further reduction of genome complexity with a factor 18.

In order to facilitate gene annotations Pacific Biosciences’s Iso-Seq™ method was used to sequence genes expressed in cotton. The full-length transcript reads also enabled the generation of a nice picture of the expression patterns of genes in leaf, stem and root tissues including the detection of tissue-specific splice variants.

The genome assembly and gene annotations were subsequently inserted into KeyGene’s proprietary genome database generating a “CottonPedia”. This cotton genome database integrates and organizes all proprietary cotton data including the greatly improved and superior genome assembly. KeyGene’s unique CottonPedia database is a powerful and user-friendly resource for breeding organizations to genetically improve cotton in applications like gene discovery and gene mapping.

“We are very pleased with the results of this collaboration, which convincingly demonstrate the advantages of Iso- Seq transcriptome analysis and long read sequencing in combination with Whole Genome Profiling in polyploid crops and the synergies between Pacific Biosciences’ platform and KeyGene’s technologies and crop expertise”, states Michiel van Eijk, Chief Scientific Officer of KeyGene. “At the PAG conference we received a lot of interesting reactions and suggestions during the KeyGene and Pacific Biosciences workshops. For instance people wondered what the quality improvement of genome assembly would be if, in order to reduce costs, WGP maps are used with 4x genome coverage (or lower) instead of 10x. We will certainly look into that to further improve our approach and to increase affordability”.

“This new method of generating reference genomes successfully leverages the combined strengths of KeyGene’s WGP and Pacific Bioscience’s Single Molecule, Real-Time (SMRT) technologies for delivering high-quality, well annotated assemblies and is particularly suited for tackling the large, polyploid genomes common to many important crop species. We are very excited about the implications of this new approach for historically difficult-to-assemble plant genomes”, states Emily Hatas, Senior AgBio Market Manager of Pacific Biosciences.

The WGP™ technology is protected by patents and patent applications owned by Keygene N.V. WGP is a trademark of Keygene N.V.