Figure 2. Sequence alignment from Clustal Omega across HBA2 and HBA1 on human chromosome 16 (Sequence data from Ensemble GRCH37).

Sequences were aligned in chromosomal order ( HBA2 [prefix 'A2_16_'] above HBA1 [prefix 'A1_16_']). The HBA2 sequences starts at the 5'-most base of the forward PCR primer (A2/3.7-F position 16:221882) in a unique region and 86 bases 5' of the homologous region with HBA1. The HBA1 sequence starts at position 16:225474 which is 319 bases from the equivalent homologous region with HBA2. The HBA2 sequence ends at position 16:223809 effectively at the end of the homologous region with HBA1, while the HBA1 sequences ends at position 16:22773 and the PCR reverse primer (3.7-R). PCR and sequencing primers ( Table 2) are highlighted (A2/3.7-R,HBA-5'-SEQ-REV, HBA-3'SEQ-FWD and 3.7-R) as are key restriction sites ¹⁰. Paralogous differences between HBA1 and HBA2 reference sequences are highlighted in green for a set of 14 positions. These were used to help identify the -α ^3.7 deletion type from Sanger sequencing. The HBA genic region is coloured and shows the transcription start site (TSS), amino-terminal methionine (ATG) and stop codons (TAA); but not separate introns and exons for clarity. The four restriction sites used to distinguish the -α ^3.7 deletion types are identified ¹⁰; the Type I breakpoint was identified as being 5' of the ApaI/IVS2 sequence; the Type II breakpoint lies between the ApaI/IVS2 and BalI restriction sites; The Type III lies between the RsaI and the 'end of HBA duplication unit' (location and identity of HBA duplication unit ²⁷). NB: The ApaI/IVS2 sequence comparison between HBA2 and HBA1 has been aligned here to clearly highlight the ApaI restriction site; it may be shown differently in other publications.