Mitochondrial DNA (mtDNA) analysis is a useful method for analyzing samples with limited or degraded DNA due to the high copy number per cell. The most common approach uses Sanger sequencing and capillary electrophoresis to analyze the two hypervariable regions (HVI and HVII) within the non-coding portion of the mitochondrial genome. However, this approach does not allow for mixture analysis, misses potentially discriminating information in the coding region, and fails if the mtDNA is too badly degraded. Next-generation sequencing (NGS) is a high throughput, massively parallel technique that allows for clonal amplification of single DNA molecules making it ideal for analyzing mixtures and heteroplasmy in mtDNA. Also, the analysis of single molecules is a more sensitive approach which can be used for analysis of limited DNA or potentially degraded samples.
This study had two aims. The first was to use the Roche 454 GS Jr to sequence the hypervariable regions of mtDNA from five comingled bone samples originating from the 20th century AD, five bone samples from the 15th century AD, and one bone sample from the 3 rd century AD. The second aim was to use probe capture and the Illumina MiSeq NGS platform to sequence the whole mitochondrial genome of artificially degraded K562 DNA, naturally degraded DNA from a modern bone sample (∼50 years old), and extremely degraded DNA from a 15th century AD bone and a 3rd century AD bone. A sub-aim was to compare a selection of small fragment removal methods following adaptor ligation to minimize sample loss at this step of library preparation.
Prior linear array and degradation qPCR results indicated that comingled remains from the 20th century AD were highly degraded. HVI & HVII sequencing using the Roche 454 was attempted because, in spite of also using long amplicons, it is a more sensitive assay. 29 comingled remains samples from the 20th century AD were amplified for 454 analysis, but only three showed successful results when viewed by gel. Those samples were sequenced by 454 along with two samples that appeared to fail amplification from this same group. All five of the comingled remains were successfully sequenced except for the HVII region of one sample, however, all were mixtures.
The five 15th century AD bone samples and 3rd century AD bone sample were highly degraded. Two of the 15th century AD samples failed sequencing entirely and another failed HVII. The 3rd century AD sample also failed HVII sequencing. There were mixtures among these samples as well, which points to the need for optimized methods of sample cleaning to remove more of the exogenous DNA present on bone samples.
A probe capture technique followed by whole genome sequencing on the Illumina Miseq was used to analyze one highly degraded bone sample from each of the 15th and 3rd centuries AD along with a naturally degraded modern bone purchased from The Bone Room in Berkeley, CA, and artificially degraded commercial K562 DNA (human chronic myelogenous leukemia cell line). Probe capture doesn’t utilize PCR amplification, therefore no specific sites are required to be intact for the assay to be successful, potentially making it a viable alternative for samples that are too degraded for PCR-based HVI and HVII analysis. The protocol calls for a small fragment removal step after adaptor ligation that could potentially remove precious sample along with the unligated adaptor, so a variety of small fragment removal methods were tested to limit the sample loss at this step of library preparation.
All of the artificially degraded samples had 100% coverage regardless of the small fragment removal method and had an average percent aligned reads of 95.59% indicating high specificity for the probe capture. Three of the four naturally degraded modern bone samples from The Bone Room had 100% coverage whereas the last sample only had a 23 bp unsequenced portion in an area with low coverage for the other samples as well. The average percent aligned reads was 49.28% indicating a capture of a substantial amount of exogenous DNA by the probe capture technique. The highly degraded ancient bone samples failed sequencing after appearing to have successfully completed library preparation, once again potentially indicating a substantial amount of exogenous DNA.
These two NGS assays were able to successfully sequence degraded bone samples. However, before they can be successful at sequencing highly degraded samples, a method of cleaning bones to remove exogenous DNA and a method of purifying extracted DNA to remove inhibitors must be optimized.
(Publisher abstract provided.)