A New Sequencing Primer and Workflow Increase 5' Resolution and Throughput on HLA Sequencing

High quality and high accuracy are the hallmarks of Sanger resequencing projects. We have developed a new sequencing primer and workflow that improves 5' sequence resolution, increases throughput, and reduces hands-on time. The novel sequencing primer chemistry produces high quality bases from base 1 on POP-7TM polymer that previously only could be resolved on the slower POP-6TM polymer. The new primer chemistry and workflow also eliminates the need for a separate PCR clean-up step. These improvements reduce the entire workflow from PCR to finished sequence data to under 5 hours, compared to 8 hours for the standard workflow. We used our enhanced sequencing primer and workflow to investigate feasibility on Human Leukocyte Antigen (HLA) polymorphisms on twelve DNA samples by using the Invitrogen SeCore® HLA-DRB1 primer set and Group Specific Sequencing Primers. Sequencing reactions generated with the traditional sequencing primer and with the new sequencing primer were electrophoresed on Applied Biosystems 3500xl Genetic Analyzer using POP-7TM polymer. For each sequencing primer, we compared 5' resolution and basecalling accuracy and quality. On average the traditional primers produced high quality readable bases by base 25 after the sequencing primer while the new primers produced high quality bases by base 5, and by base 1 in many cases. Because of improved resolution, basecalling accuracy was increased. This simplified process without a separate PCR clean-up step reduced the overall workflow time by 40%. For HLA genes, obtaining readable sequence within 5 bases of the primer offers improved polymorphism detection and more efficient use of allele specific sequencing primers for heterozygous ambiguity resolution. In conclusion, the novel primer chemistry and workflow generates data superior in quality relative to other currently used solutions and offers significant time savings as well. Specific applications of this product are under development and not intended for clinical use.