DEVELOPMENT OF A FULL MITOCHONDRIAL GENOME SPECIFIC TARGET-ENRICHED LIBRARY FOR NEXT GENERATION SEQUENCING APPLICATIONS

Purpose: The purpose of this study is to develop a target-enriched full mitochondrial genome library for a set of database samples from Easter Island to be used for downstream next generation sequencing applications. The samples did not amplify with a previously-developed polymerase chain reaction (PCR) primer multiplex that was expected to amplify overlapping mitochondrial DNA (mtDNA) fragments of approximately 1,500 -3,000 base pairs (bp) in length. Possible explanations of this unsuccessful amplification are either the sample quality is low or the FTATM cards on which the samples are stored prevents amplification of very large fragments of DNA. It is hypothesized that successful amplification of the samples can be achieved using a multiplex of PCR primers with a smaller target amplicon size. Methods: Twenty-four primer pairs developed by Rieder et al. were obtained that amplify the entire mitochondrial genome in overlapping fragments approximately 900 bp in length. The primers were tested in single-plex reactions first to ensure each primer pair was amplifying as expected. PCR results were observed and analyzed using the Agilent 2100 Bioanalyzer, an instrument that uses electrophoresis to separate, size and quantify fragments of DNA in a sample. Once all the fragments covering the entire mtDNA were amplified in single PCR reactions, 2 multiplex reactions each with 12 primer pairs were developed so that only 2 reactions were needed to generate the whole mtDNA amplicon pool instead of 24. An amplicon ladder for each of the two multiplexes was created by compiling the amplicons from the single-plex reactions. These ladders were used for comparison with the multiplex reaction results to ensure all fragments successfully amplified and were at balanced concentrations in the multiplex PCR reactions. Results: Electropherograms displaying the size and concentration of the DNA fragments amplified in each PCR reaction were generated by the Agilent 2100 Bioanalyzer. The electropherograms of the multiplex reactions closely resembled the electropherograms of the amplicon ladders. All of the expected amplicons were present in the multiplexed reactions and at relatively balanced concentrations. Conclusions: Successful whole mtDNA amplification was accomplished by using PCR primers with smaller target amplicons. These newly developed amplicon pools can now be used to create next generation sequencing libraries. The multiplex primer reactions developed can be used for similarly challenged samples in the future.