Strategies for sequencing the HAS1 gene segments. Described are 2 strategies used to amplify the HAS1 gene segments. Overlapping reverse and forward primers were designed to anneal with exons and introns of the HAS1 gene to identify genetic variations that may contribute to aberrant HAS1 splicing in patients with WM. Minigene sequencing in strategy 2 was used to determine whether the recurrent mutations were clustered. gDNA PCR and cloning were carried out as described in “Methods.” We picked 24 to 48 subclones to screen and sequence inserts (gDNA PCR product of the HAS1 gene segment) in the TOPO TA plasmid using the appropriate primer sets for each segment. While using the first strategy of amplifying HAS1 gene segments from the patients or HDs, we cloned 7 or 3 segments from the exon 3 to exon 5 region of the HAS1 gene using A or B primer sets, respectively. For each segment, 3 to 10 positive subclones were selected, and, for each cell subset, more than 50 plasmids were isolated and sequenced both directions using M13 and T7 sequencing primers. Using strategy 2, we cloned 30 HAS1 minigene plasmids from MM and WM, and 33 minigenes from B-CLL and MGUS. B-CLL minigenes encompassed only intron 4. MGUS minigenes encompassed exon 3 to exon 5. Each plasmid was sequenced using overlapping HAS1 gene-specific A and B primer sets. Because we used overlapping primers either in gDNA PCR (strategy 1) or for sequencing (strategy 2), we analyzed 50 to 60 sequencing reactions for exon-intron spanning segments of the HAS1 gene. The HAS1 gene segments of 2 HDs (B and T cells) were sequenced using strategy 1. The genetic variations identified in patients with MM and WM were assessed, based on a total of 4119 sequencing reactions.