Table 3 Strengths, weakness, improvements and new opportunities of the CRISPR-Cas9 sequencing enrichment strategy described in this study
Strengths | Weakness | Improvements | New opportunities |
|---|---|---|---|
The design of crRNAs does not necessarily require a reference genome Pooling the crRNAs and barcoded DNAs results in a simple and cost-effective method Polymorphisms can be extracted and phased with high efficiency from genome alignments and/or de novo sequences The method is computationally inexpensive | The search for polymorphisms in the de novo sequences might require the manual isolation and comparison of contigs ONT technology is prone to sequence errors in homopolymer regions [48] The Cas9 digestion with a single crRNA pool cannot produce overlapping fragments | Digesting the DNAs with sub-pools of crRNA might improve the region assembly, at the expenses of simplicity and cost-effectiveness The use of samples homozygous for the target region might increase the efficiency of polymorphism discovery and sequence scaffolding | Identification of methylation variants between the pooled samples in the target region [23, 25] Enrichment of genes scattered in more than one region of interest, such as the enrichment of specific gene or transposon families[41] |