Table 4. Target nucleotide mutation frequency in DGYW/WRCH or WA/TW mutation hotspots within framework regions (FR) and complementarity determining regions (CDR).

[DGYW/WRCH (G:C is the mutable position; D = A/G/T, Y = C/T, W = A/T, R = A/G, and H = T/C/A); WA/TW (A:T is the mutable position; W = A/T); ‘ALL’ refers to nucleotides within a hotspot motif; ‘All other’ refers to nucleotides outside a hotspot motif]

Mutated base	Hotspot motif	Region	Total nucleotides	Observed # Mutations	Expected # Mutations	Mutation freq (%)	χ2 p	T-test P
G/C	DGYW/WRCH	FR	927	56	18.16	6.04	0.0000	0.0267
		CDR	400	35	26.22	8.75
		ALL	1327	91	37.52	6.86	0.0000
	Outside Motif		4402	71	124.48	1.61
A/T	WA/TW	FR	2531	54	36.07	2.13	0.0015	0.2248
		CDR	400	26	21.12	4.03
		ALL	2931	80	55.97	2.52	0.0000
	Outside Motif		3690	41	65.03	1.11

*T-test analysis was used to compare mutations within hotspot motifs to those outside hotspot motifs. Mutations to G and C nucleotides occurred significantly more often within DGYW/WRCH motifs than outside these motifs, while mutations to A and T nucleotides showed no preference for WA/TW motifs.

†χ2 analysis was used to compare observed and expected numbers of mutations between FR and CDR regions and between mutations inside and outside hotspot motifs. More mutations to all nucleotides occurred within hotspots than outside hotspots, and significantly more mutations occurred to nucleotides within CDRs than FRs.