The LAM-HTGTS platform
High-throughput Genome-wide Translocation Sequencing (HTGTS) was developed originally to identify recurrent “prey” DSBs genome-wide using minimal selective forces (Chiarle et al., 2011). A second generation of the assay was developed, involving linear amplification (LAM) with a single primer, ligation of the single DNA strand using thermodynamically stabilizing bridge adapters (LAM-HTGTS) (Frock et al., 2015; Hu et al., 2016) and a complete overhaul of the junction analysis pipeline to enable single nucleotide resolution measures of translocation bait/prey junctions. LAM-HTGTS has served as the basis for several downstream applications including antigen receptor repertoires (Lin et al., 2016), DNA:DNA interactions (Jain et al., 2018), and comprehensive end-joining measures in non-dividing cells (Liang et al., 2021).
A long-term focus is to further develop/adapt this platform for basic research and translational studies
Key HTGTS feature: Readily detectable frequent DSBs
From on-target (red) and off-target (blue/green) Cas9:gRNA endonuclease bait DSBs (Frock et al., 2015). Nearly any recurrent DSB site can be used.
Universal bait DSB approach to evaluate endonuclease specificity & efficacy
Orthogonal HTGTS: using different species Cas9 bait / prey DSBs (S.aureus - Sa / S.pyogenes - Sp) to compare engineered SpCas9 variant, eSpCas9(1.1). Collaboration with the Gonçalves group at Leiden University (Wang et al., 2021; Chen et al., 2020).