Custom PureParget panels are not supported by PacBio
Custom PureTarget panels are not officially supported by PacBio. Users must design and order their guide RNAs and optimize their custom panels accordingly. PacBio can offer limited guidance on guide RNA design software and strategies for optimizing custom panels. We recommend first demonstrating success on the PureTarget repeat expansion panel using supported sample types before adding new guide RNAs or testing a custom set of guides.
Adding more validated repeat expansion targets: This “flavor” of customization is relatively low risk. Internally, we have demonstrated success in adding up to 5 pairs of guides for additional repeat expansion targets. The design strategy is the same as the fixed 20 gene panel: design guides ~2 kb upstream and downstream of the repeat expansion region with fragments ranging from 3-5 kb.
Adding non-repeat expansion targets: Internally, we have demonstrated success in adding a pair of guides to target a full-length gene with fragment size of ~5 kb, similar in size to the PureTarget repeat expansion panel. Please consult with your field application bioinformatics support scientist for the recommended variant calling tools for your targets.
All other designs like capturing fragments longer than 5 kb or using a tiled design are untested and we do not have any recommendations on design or optimization.
A software tool we recommend for designing guide RNAs is Custom Alt-R™ CRISPR-Cas9 guide RNA from Integrated DNA Technologies (IDT) available here: https://www.idtdna.com/site/order/oligoentry/index/crisprcustom.
We recommend ordering SygRNAs (Synthetic Single Guide RNAs) from MilliporeSigma or sgRNA from IDT rather than the two-part targeting system composed of a separate tracrRNA and crRNA. This way, you only need to order one gRNA and the preparation is simplified (there is no annealing of the two molecules).