Read Cloud Wrangling: Targeted Assemblies from Linked Reads for Genome Editing Applications

Genome editing is poised to have a big impact on agricultural biotechnology. Yet, in order to modify the genome of an elite plant line, the sequence underlying a desired target gene (and the surrounding region) must be known.... [ view full abstract ]

Genome editing is poised to have a big impact on agricultural biotechnology. Yet, in order to modify the genome of an elite plant line, the sequence underlying a desired target gene (and the surrounding region) must be known. For this application, long range contiguity is not as critical as assembly accuracy/completeness across the target region and the timeline for obtaining the desired sequence. The rapidly evolving landscape of short/long read sequence technologies offers a wide-range of approaches for obtaining a target sequence, such as WGS re-sequencing, amplicons, sequence capture, BACs, and whole-genome assembly. The overall utility of these methods is generally proportional to the cost, time, and effort associated with generating and assembling the data. Thus, there is need for a new sequencing approach that is both rapid and cost-effective, yet offers robust assemblies suitable for genome-editing applications. Linked read technology (or read clouds) has the potential to fill this application gap by combining aspects of short and long reads via high-throughput sequencing of barcoded paired-end libraries derived from long molecules. Read clouds derived from a given region can be readily identified on the basis of barcodes and specifically assembled to quickly obtain a ~20-100 kb assembly spanning a genome editing target gene. This approach for “read cloud wrangling” using linked reads to support genome editing applications in plants will be discussed.