Easily isolate long DNA fragments that overcome challenges of sequencing large and complex plant genomes
The 5.3-Gb genome of barley includes several gene clusters involved in the biosynthesis of natural products. Barley has a complex genome with a large proportion of transposable elements with repetitive sequences. Most varieties do not have a reference genome.
In this experiment, we used Xdrop to enrich a specific genomic region to enable in-depth sequence elucidation. Download the application note for full details of how this technology helps reveal gene cluster structure even when only limited knowledge about the variety-specific sequence is available.
Angiosperm genome sizes span 4 orders of magnitude2
Percent of genome consisting of repeat sequences and transposable elements increases with genome size2
Most published plant genomes are 15 % incomplete due to high copy number repeats2
We used Xdrop to investigate the gene cluster that synthesizes falcarindiol in the tomato plant. Metabolic and mRNA expression analyses published by researchers at Stanford University had identified 8 candidate genes of the cluster.3 Alas, only 2 were partially matched in the reference genome and an attempted WGS using Oxford Nanopore Technology generated 4 Gb of data but only 3 reads in the region of interest! With Xdrop, we achieved 980-fold enrichment of a 100 kb region around one of the identified genes with only 0,39 Gb of data. Sequencing fold coverage reached up to 400x in the cluster region, and allowed for recontructing the genomic region using de novo assembly.
1 Schatz, M.C., et al. 2012. Current challenges in de novo plant genome sequencing and assembly. Genome Biol. 13: 243.2 Michael, T.P. 2014. Plant genome size variation: bloating and purging DNA. Brief. Funct. Genomics 13: 308.3 Jeon, J.E., et al. 2020. A pathogen-responsive gene cluster for highly modified fatty acids in tomato. Cell 180: 176.
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