De novo Sequencing of the Vitis vinifera cv. Flame Seedless Genome using Single Pass Library Preparation from Swift Biosciences

Single Molecule Real-Time (SMRT) sequencing provides advantages to the sequencing of complex genomes. The long reads generated are superior for resolving complex genomic regions and provide highly contiguous de novo assemblies. Current SMRTbell libraries generate average read lengths of 10-15kb. However, genomes with higher repeat content or DNA that is difficult to extract can result in yield and read lengths below the expected values. Recently, Swift Biosciences has developed a kit for SMRT sequencing that does not require the use of SMRTbells. This approach reduces library preparation the time to 24 hours while also simplifying the approach. The simplified protocol results in longer read lengths from less starting material. These improvements are particularly striking in hard to sequence genomes.

We employed this approach in sequencing the heterozygous and repetitive genome of Vitis vinifera cv. ‘Flame seedless’ genome; one of the most widely planted table grape varieties. A contiguous assembly of this cultivar will provide insight into traits that differentiate seedless table grapes from wine grapes and reveal important structural elements. Compared to the SMRTbell construction, the single pass kit resulted in twice the yield per SMRTcell. Additionally, the N50 increased from 9,711bp to 12,289bp while the proportion of reads >10kb to 39.4% compared to 15% in the original preparation. Furthermore, the longest read generated by the Swift prep was 73,088bp compared to 59,854bp from the SMRTbell preparation. This increased yield and N50 is essential to creating a high-quality reference genome using long-read sequencing with less DNA at lower cost.