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11.01.2017 | Original Article | Ausgabe 4/2017

International Journal of Legal Medicine 4/2017

MPS analysis of the mtDNA hypervariable regions on the MiSeq with improved enrichment

Zeitschrift:
International Journal of Legal Medicine > Ausgabe 4/2017
Autoren:
Mitchell M. Holland, Laura A. Wilson, Sarah Copeland, Gloria Dimick, Charity A. Holland, Robert Bever, Jennifer A. McElhoe
Wichtige Hinweise

Electronic supplementary material

The online version of this article (doi:10.​1007/​s00414-017-1530-9) contains supplementary material, which is available to authorized users.

Abstract

The non-coding displacement (D) loop of the human mitochondrial (mt) genome contains two hypervariable regions known as HVR1 and HVR2 that are most often analyzed by forensic DNA laboratories. The massively parallel sequencing (MPS) protocol from Illumina (Human mtDNA D-Loop Hypervariable Region protocol) utilizes four sets of established PCR primer pairs for the initial amplification (enrichment) step that span the hypervariable regions. Transposase adapted (TA) sequences are attached to the 5′-end of each primer, allowing for effective library preparation prior to analysis on the MiSeq, and AmpliTaq Gold DNA polymerase is the enzyme recommended for amplification. The amplification conditions were modified by replacing AmpliTaq Gold with TaKaRa Ex Taq® HS, along with an enhanced PCR buffer system. The resulting method was compared to the recommended protocol and to a conventional non-MPS approach used in an operating forensic DNA laboratory. The modified amplification conditions gave equivalent or improved results, including when amplifying low amounts of DNA template from hair shafts which are a routine evidence type in forensic mtDNA cases. Amplification products were successfully sequenced using an MPS approach, addressing sensitivity of library preparation, evaluation of precision and accuracy through repeatability and reproducibility, and mixture studies. These findings provide forensic laboratories with a robust and improved enrichment method as they begin to implement the D-loop protocol from Illumina. Given that Ex Taq® HS is a proofreading enzyme, using this approach should allow for improved analysis of low-level mtDNA heteroplasmy.

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Zusatzmaterial
Supplemental Fig S1 Comparison of amplification results associated with the four individual primer sets, as shown cumulatively in Fig. 1; primer set 1 (A), 2 (B), 3 (C) and 4 (D). Quantification results for amplification reactions of buccal extracts, comparing the Illumina and PSU protocols. Template amounts are related to nuclear (n) DNA quantities. Qubit-based quantification results were normalized to a 50 uL PCR reaction volume. The experiments were performed at Penn State. Error bars represent standard error of the mean (SEM), with an n value of 4 per primer set. (PDF 175 kb)
Supplemental Fig S2 Comparison of amplification results associated with the four individual primer sets, as shown described in Fig. S1. The experiments were performed at Mitotyping Technologies. Error bars represent standard error of the mean (SEM), with an n value of 4 per primer set; “*” means p < 0.05 and “**” means p ≤ 0.01. (PDF 151 kb)
414_2017_1530_MOESM2_ESM.pdf
Supplemental Fig S3 Comparison of amplification results when using conventional or transposase adapted (TA) PCR primers with the PSU protocol. Error bars represent standard error of the mean (SEM), with an n value of 16 (4 primer sets X 4 amplifications); “*” means p < 0.05. (PDF 48 kb)
414_2017_1530_MOESM3_ESM.pdf
Supplemental Fig S4 Comparison of amplification results for the PSU protocol (Ex Taq® HS DNA polymerase and associated buffer system) at different primer concentrations; 400 nM and 800 nM. Buccal DNA template amounts are 5 pgs (A) and 500 pgs (B). Qubit-based quantification results were normalized to a 50 uL PCR reaction volume. Error bars represent standard error of the mean (SEM), with an n value of 4 per primer set; “**” means p < 0.01. (PDF 149 kb)
414_2017_1530_MOESM4_ESM.pdf
Supplemental Fig S5 (A) Comparison of amplification results on DNA extracts from human hair shafts when using the conventional and PSU protocols; the use of conventional primers and AmpliTaq Gold DNA polymerase (conventional) or TA primers and Ex Taq® HS DNA polymerase (PSU), with their associated buffer systems. Hair shaft DNA template amounts were unknown. Instead, 10 uL of extract was used in each amplification reaction. Qubit-based quantification results were normalized to a 50 uL PCR reaction volume. Error bars represent standard error of the mean (SEM), with an n value of 32 (4 hairs X 4 primer sets X 2 amplifications); “*” means p = 0.105. (B) Comparison of amplification results associated with the four individual primer sets. Quantification results for amplification reactions of hair shaft extracts, comparing the conventional and PSU protocols; the use of conventional primers and AmpliTaq Gold DNA polymerase (conventional) or TA primers and Ex Taq® HS DNA polymerase (PSU), with their associated buffer systems. Template amount was not determined. Instead, 10 uL of extract was used in each amplification reaction. Qubit-based quantification results were normalized to a 50 uL PCR reaction volume. Error bars represent standard error of the mean (SEM), with an n value of 8 per primer set. (PDF 85 kb)
414_2017_1530_MOESM5_ESM.pdf
Supplemental Fig S6 A screen shot of the Mix 2 data set in the GeneMarker® HTP software. Reads are aligned in a “packed” manner to maximize the space filling aspect of the pileup. The region of sequence in the viewing window ranges from approximately np 15,998 to 16,418, including nps 16,069, 16,126, 16,319, and 16,368, and is reflective of the pileup from approximately read 2879 to 2919. It is clear that the region of the pileup for np 16,126 has a visible gap associated with the collective process of tagmentation, read filtering, and read alignment. (PDF 217 kb)
414_2017_1530_MOESM6_ESM.pdf
Supplemental Table S1 Standard deviations for all data associated with the enrichment study. (DOCX 30 kb)
414_2017_1530_MOESM7_ESM.docx
Supplemental Table S2 Data for the two mixture series. (XLSX 52 kb)
414_2017_1530_MOESM8_ESM.xlsx
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