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Evaluation of the ForenSeq mtDNA Whole Genome Kit for massively parallel sequencing of mitochondrial genomes

Open AccessPublished:October 25, 2022DOI:https://doi.org/10.1016/j.fsigss.2022.10.065

      Abstract

      The performance of Verogen’s ForenSeq mtDNA Whole Genome Kit and its possible application in a forensic setting was evaluated. We carried out three sequencing runs that included 1) a dilution series of two different samples (200–6.25 pg input nuclear DNA), 2) casework samples, and 3) hair, blood, and buccal samples from the same individual. For samples described in 1), the method was tested using different reagent volume in the PCR and library building (recommended volume or half-volume). Reference samples were analysed in duplicates, and positive and negative controls were included in every run. All mtDNA profiles were known from previous genotyping with the Precision ID mtDNA Whole Genome Panel or using long range PCR and the MiSeq instrument. The average read depth per sample was > 1200x in all samples, although some regions had read depth 50x. Using half volume of reagents did not compromise the quality of the results and can be a cost-effective alternative for reference samples. For an analytical threshold of 10%, complete concordance was observed between the duplicates and previous results. Heteroplasmy was consistent with prior observations although frequencies varied up to 10% compared to previous results.

      Keywords

      1. Introduction

      Massively parallel sequencing has become accessible as a fast and cost-effective method for sequencing of the whole mtDNA genome [
      • Børsting C.
      • Morling N.
      Next generation sequencing and its applications in forensic genetics.
      ,
      • Parson W.
      • Strobl C.
      • Huber G.
      • et al.
      Evaluation of next generation mtGenome sequencing using the ion torrent personal genome machine (PGM).
      ,
      • King J.L.
      • LaRue B.L.
      • Novroski N.M.
      • et al.
      High-quality and high-throughput massively parallel sequencing of the human mitochondrial genome using the Illumina MiSeq.
      ]. The ForenSeq™ mtDNA Whole Genome Kit amplifies the entire mtDNA genome in two multiplex PCRs covering 245 amplicons with an average amplicon size of 131 bps. The kit is optimised for 100 pg DNA input (50 pg in two separate primer pools), and libraries are sequenced on an MiSeq FGx® platform.
      This study aimed to evaluate the performance of the ForenSeq™ mtDNA Whole Genome Kit and its potential application in a forensic setting. The method was tested using different DNA inputs, and reagent volumes in the PCR and library building (recommended volume or half-volume). Positive (HL-60) and negative controls were included in each run.

      2. Material and methods

      2.1 Sensitivity study and reagent volume study

      Previously extracted samples from two individuals, A and B, were quantified with a Qubit® 3.0 Fluorometer using the Qubit dsDNA HS Assay Kit (Thermo Fisher Scientific) according to the manufacturer’s instructions. Samples were diluted to 200 – 100 – 25 – 6.25 pg/µL and analysed in duplicates using either the recommended reagent volume or half of the recommended reagent volume for amplification and library building.

      2.2 Casework samples

      DNA was below the threshold of detection of the Qubit® 3.0 Fluorometer. Maximum volume was used for the PCR (3 µL per primer pool).

      2.3 Hair and reference samples

      One buccal swab, one blood sample on FTA card, and hair shafts were collected from two individuals. Buccal swabs and blood samples were extracted with the standard EZ-1 protocol (Qiagen) and quantified with Qubit. Hair sample preparation and extraction was according to [
      • Brandhagen M.D.
      • Loreille O.
      • Irwin J.A.
      Fragmented nuclear DNA is the predominant genetic material in human hair shafts.
      ]).
      The study follows the policy from the Science Ethics Committees for the Capital Region (De Videnskabsetiske Komitéer for Region Hovedstaden) regarding method development/validation studies in non-health related projects and complies with the rules of general data protection regulation. Casework samples are registered at the University of Copenhagen’s general records of processing activities (004–0065/21–7000).

      2.4 Library building, sequencing, and data analysis

      Libraries were manually prepared according to the manufacturer’s protocol (Verogen). Sixteen samples including positive (HL-60) and negative controls were loaded onto standard MiSeq FGx® (Verogen) flow cells. Samples were sequenced on the MiSeq FGx® (Verogen) instrument. Data analysis was performed using the ForenSeq Universal Analysis Software (UAS) 2.0 module (Verogen). BAM files were manually inspected using the IGV software to confirm the polymorphisms reported by the UAS.

      3. Results and discussion

      3.1 Sensitivity study and half reagent volume study

      Amplification was successful even in samples with low input of nuclear DNA (6.25 pg) although for lower inputs, some positions had read depths below 100x, and most 6.25 pg samples had minimum read depths below 50x (Fig. 1). Similar results were obtained for the four casework samples, with only one sample presenting minimum read depth < 50x. Using half of the recommended volumes did not compromise the quality of the results, in fact, minimum read depths were > 100x for these samples (Fig. 1).
      Fig. 1
      Fig. 1Maximum, average (red line) and minimum read depth per sample using full and half reagent volumes for amplification and library preparation.
      Some regions occasionally failed to be amplified, especially for 6.25 and 25 pg inputs. These regions were: 470–519, 6774–6810, 10,203–10,279, 13,657–13,701, that had low relative read depths in general.
      Poly C regions (303–315 and 16,180–16,193) and repetitive regions (514–524) should be carefully inspected or excluded from the analysis.

      3.2 Concordance

      Most positions were consistent with previous results and among the different tissues, when considering a variant calling threshold of 10%. However, three of the tested samples had missing data in region 470–519, and in two samples, it had consequences on the identified haplotype. Some samples had 1–2 variants, that were not reproduced in other samples or tissues from the same individual. One 309.1 C variant in Sample A [
      • Pereira V.
      • Longobardi A.
      • Børsting C.
      Sequencing of mitochondrial genomes using the precision ID mtDNA whole genome panel.
      ] was not reproduced in any of the ForenSeq™ mtDNA Whole Genome Kit analyses.

      3.3 Heteroplasmy

      Previously reported heteroplasmic positions were confirmed. Heteroplasmic positions reported with both the ForenSeq™ mtDNA Whole Genome Kit and the Precision ID mtDNA Whole Genome Panel are presented in Fig. 2. Most variant frequencies were equal (or very similar). However, previously identified positions with heteroplasmy (5752, 7471, 8254, 8256, 10,885) in the casework samples [
      • Pereira V.
      • Longobardi A.
      • Børsting C.
      Sequencing of mitochondrial genomes using the precision ID mtDNA whole genome panel.
      ] were not reproduced using the ForenSeq™ mtDNA Whole Genome Kit analyses (data not shown).
      Fig. 2
      Fig. 2Heteroplasmic positions reported with both chemistries. The most frequent variant is marked in bold.

      4. Conclusion

      • The average read depth per sample was > 1200x in all samples, although some regions had read depth < 50x.
      • Using half volume of reagents did not compromise the quality of the results and can be a cost-effective alternative for reference samples.
      • The Universal Analysis Software (UAS) reports variants above 6%. When the interpretation threshold was set to 10%, complete concordance was observed between the duplicates and previous results (excluding PolyC regions 303–315 and 16,180–16,193, and the repetitive region 514–524).
      • Heteroplasmy reported with both chemistries was consistent, although frequencies varied up to 10% compared to previous results.

      Acknowledgements

      The authors wish to thank Nadia Jochumsen for technical assistance.

      Conflict of interest

      None.

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