GUIDE-seq is an experimental method to detect off-target cleavages caused during CRISPR\/Cas editing.See publication https:\/\/doi.org\/10.1038\/nbt.3117 for details about the method, the protocol used, and the analysis performed.We identified several areas of improvement in the published protocol for GUIDE-seq NGS library preparation and developed a simplified protocol that is easier to perform & less expensive. The new protocol is also load-and-go compatible with standard sequencing runs on all Illumina instruments (using the kit SBS reagents & default instrument settings) compared to the NBT protocol that requires the libraries to be run on a MiSeq with some tweaks to the software configuration.Notable changes:1. Moved the UMI (Unique Molecular Identifier) from the Y-adapter to be inline with the insert. UMI is now sequenced as part of the sequencing read (R2 on a paired-end run). This eliminates the need to export index reads from the MiSeq.2. Removed the sample index from the Y-adapter and changed the PCR scheme. Now, only a single adapter is required for ligation to any number of samples, PCR1 is for enrichment of the dsOligo - genome junction, PCR2 is to add sample-specific indexes to the enriched amplicons. See oligo designs for further information.3. Replaced sonication based shearing followed by end-repair with a one-pot, one-step enzymatic fragmentation + end-repair kit from New England Biolabs.