Examining Effects of PCR Conditions on mtDNA Heteroplasmy Detection Using Next Generation Sequencing
Open Access
- Author:
- Rothwell, Lauren Elizabeth
- Area of Honors:
- Forensic Science
- Degree:
- Bachelor of Science
- Document Type:
- Thesis
- Thesis Supervisors:
- Dr. Mitchell Mark Holland, Thesis Supervisor
Dr. Mitchell Mark Holland, Thesis Honors Advisor
Ronald D Porter, Faculty Reader - Keywords:
- forensic science
DNA
heteroplasmy
pyrosequencing
chimeric sequences - Abstract:
- ABSTRACT Aim: To examine the effects of varying PCR conditions on the detection and differentiation of low level mtDNA heteroplasmy and the artificial production of chimeric sequences when performing deep pyrosequencing. Methods: A hypervariable segment (HV1) of the mtDNA control region was analyzed from several individuals using the 454 GS Junior instrument. Similar to previous research experiments, mock mixtures of two individuals were analyzed to evaluate low level heteroplasmy and deconvolute mtDNA mixtures. During previous experiments (Holland et al 2011), the mtDNA mixtures showed chimeric sequences, or sequences that contained the known polymorphisms of both individuals in the mixture. The current study used alterations in first round amplification conditions to reduce or eliminate chimeric sequences in order to more accurately examine low level heteroplasmic variants. Amplicon sequencing was performed on PCR products generated with fusion primers that included multiplex identifiers (MID) and adaptors for pyrosequencing. Data analysis was performed using NextGENe® software. Results: Dilution experiments showed a decrease in observed chimeric sequences at 10x dilution. Using input DNA concentrations less than the protocol’s optimal range showed higher percentages of chimeric reads than 1:10 dilution. Decreasing input DNA to these concentrations resulted in little correlation of dilution and observed chimeric minor reads. Increasing the first round PCR extension time by one minute resulted in decreased chimeric reads at both 28 cycles (protocol) and 38 cycles (hair shaft protocol). Notable increases in chimeric reads were seen at 38 cycles. Conclusions: Changing the first round PCR settings such as input DNA concentrations, cycle number and extension times do have an effect on downstream mtDNA sequencing results when using NGS pyrosequencing technology. While increasing cycle number to 38 cycles increases the observance of chimeric minor sequences in a mixture, diluting tenfold and increasing extension time by at least 1 minute decreases total chimeric reads. The highest observed chimeric percentage reached 30%, meaning 30% of the total minor component sequences in the mixture sample showed polymorphisms from both contributors of the mixture. This percentage must be reduced in order to properly analyze minor contributor sequences, especially when low level heteroplasmy is observed and could provide additional information for that sample.