AB118. Validation of next generation sequencing by Sanger sequencing

Background and objective: Development of the next generation sequencing (NGS) platform was driven by the completion of the Human Genome Project in 2003. With the availability of NGS, the time taken for sequencing of humongous genomic regions was greatly reduced and data generated per unit DNA was also significantly increased. Though the cost to use NGS in a clinically setting is far from ideal, economically speaking, there is a significant decrease in the average cost per sequenced base. To validate findings of NGS on mutation detected for FBN1, TGFBR2, RAF1, RTEL1, LMNA, MID2, KCNK9, DMD, SMARCA2 and IQSEC2 by using gold standard, Sanger Sequencing.

Methods: The coordinate of the mutation identified by NGS was used to retrieve the adjacent genomic sequence in UCSC Genome Browser (Available from URL: https://genome.ucsc.edu/). Targeted primers were designed with Primer 3 software (Available from URL: http://primer3.ut.ee/) based on the genomic sequence obtained from UCSC. The following step involves the optimization of a Polymerase Chain Reaction (PCR) with the designed primers to amplify the desired DNA template for the targeted region. Upon optimization, the template is purified and subjected to dye terminator sequencing to generate multiple DNA fragments of varying sizes. Lastly, the DNA fragments will be purified and analysed with an automated sequencer. The sequencer separates the DNA fragments based on their size by carrying out capillary electrophoresis.

Results: A total of 28 cases were validated with Sanger sequencing. Of them, 25 (89.3%) cases concur with the findings from NGS and 3 (10.7%) cases were false-positive calls.

Conclusions: NGS shows promise in the future molecular diagnostic regime, however, at the present moment, it needs to be done concurrently with Sanger sequencing for clinical applications.

Keywords: Next generation sequencing (NGS); Sanger sequencing