Extended Data Figure 2: Genome-wide translocation junctions lack orientation bias; statistical analyses for experimental replicates orientation-biased joining between I-SceI break in place of Sα and AID-initiated Sμ breaks in CH12F3 cells.

a, b, Circos plots for translocation junctions across the whole genome from 3′-broken end (a, n = 4) or 5′-broken end (b, n = 3) HTGTS with anti-CD40/IL4 stimulated ΔSμ^2×I/ΔSγ1^2×I B cells. c, d, Bar graphs depicting genome-wide percentage of junctions from pooled 3′- and 5′-broken end libraries plotted separately in − or + orientations. Error bars are s.d. e, Joining from ΔSγ1^2×I 3′-broken end to AID off-target DSBs in Il4ra gene on chromosome 7. f, Bar graph showing the number of junctions (average ± s.d.) recovered from Igh^I-96k AID^−/− 3′-broken end HTGTS libraries (n = 3) at the break site and the upstream Sμ^1×I prey break as a percentage of the total number of junctions mapped to the 200 kb Igh constant region. Right panel shows the percentage of junctions mapping at Sμ^1×I (average ± s.d.) over the total Igh junctions that are mapped in the deletion (Del) or inversional (Inv) orientation. The numbers above the bar graph (average ± s.d.) denote the ratio of deletional to inversional junctions. g, Percentage of junctions (average ± s.d.) recovered from the Igh^I-96kAID^−/− 5′-broken end HTGTS libraries (n = 3). h, Percentage of junctions (average ± s.d.) recovered from the ΔSμ^2×I/ΔSγ1^2×I 3′-broken end libraries (n = 4). i, Percentage of junctions (average ± s.d.) recovered from the ΔSμ^2×I/ΔSγ1^2×I 5′-broken end libraries (n = 3). j, Percentage of junctions (average ± s.d.) recovered from the wild-type ΔSγ1^2×I 3′-broken end libraries (n = 3). k, Percentage of junctions (average ± s.d.) recovered from the ΔSα^1×I CH12F3 3′-broken end libraries (n = 3) and ΔSα^1×I Sμ(INV) CH12F3 cells 3′-broken end libraries (n = 3). l, Bar graphs depicting percentage of trans junctions mapping to Sμ in − and + orientations from libraries of ΔSμ^2×I/ΔSγ1^2×I B cells (n = 3) cloning from ΔSγ1^2×I 3′-broken ends. m, Bar graphs depicting percentage of trans junctions mapping to Sμ in − and + orientations and to Sε in − and + orientations from libraries of c-myc^25×I 5′-broken ends (n = 3). n, o, HTGTS library analyses of ΔSα^1×I CH12F3 cells stimulated with anti-CD40, IL4 and TGFβ and nucleofected with I-Sce1 expression plasmid. Cells were harvested on day 3 post-stimulation for 3′-broken ends (n, n = 6) and 5′-broken ends (o, n = 6) libraries. p, 3′- and 5′-broken end libraries are normalized with ‘symmetric junctions’ (see Supplementary Information). q, Bar graph showing percentage of junctions from ΔSα^1×I CH12F3 cells (n = 6) from 3′- and 5′-broken end primers. For detailed legends and further discussion refer to the Supplementary Information.