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Effects of the Ion PGM™ Hi-Q™ sequencing chemistry on sequence data quality

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Abstract

Massively parallel sequencing (MPS) offers substantial improvements over current forensic DNA typing methodologies such as increased resolution, scalability, and throughput. The Ion PGM™ is a promising MPS platform for analysis of forensic biological evidence. The system employs a sequencing-by-synthesis chemistry on a semiconductor chip that measures a pH change due to the release of hydrogen ions as nucleotides are incorporated into the growing DNA strands. However, implementation of MPS into forensic laboratories requires a robust chemistry. Ion Torrent’s Hi-Q™ Sequencing Chemistry was evaluated to determine if it could improve on the quality of the generated sequence data in association with selected genetic marker targets. The whole mitochondrial genome and the HID-Ion STR 10-plex panel were sequenced on the Ion PGM™ system with the Ion PGM™ Sequencing 400 Kit and the Ion PGM™ Hi-Q™ Sequencing Kit. Concordance, coverage, strand balance, noise, and deletion ratios were assessed in evaluating the performance of the Ion PGM™ Hi-Q™ Sequencing Kit. The results indicate that reliable, accurate data are generated and that sequencing through homopolymeric regions can be improved with the use of Ion Torrent’s Hi-Q™ Sequencing Chemistry. Overall, the quality of the generated sequencing data supports the potential for use of the Ion PGM™ in forensic genetic laboratories.

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Acknowledgments

We thank Thermo Fisher Scientific for providing reagents necessary to complete this study and Robert Lagace, Joseph Chang, Sharon Wootton, and Chien-Wei Chang, specifically, for their necessary technical expertise. We also thank Monika Stoljarova for her technical expertise and work in developing background knowledge over the potential causes of noise seen when sequencing the mitochondrial genome with MPS.

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Authors and Affiliations

  1. Institute of Applied Genetics, Department of Molecular and Medical Genetics, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., CBH-250, Fort Worth, TX, 76107, USA

    Jennifer D. Churchill, Jonathan L. King, Ranajit Chakraborty & Bruce Budowle

  2. Center of Excellence in Genomic Medicine Research (CEGMR), King Abdulaziz University, Jeddah, Saudi Arabia

    Bruce Budowle

Authors
  1. Jennifer D. Churchill
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  2. Jonathan L. King
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  3. Ranajit Chakraborty
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  4. Bruce Budowle
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Corresponding author

Correspondence to Jennifer D. Churchill.

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The authors declare that they have no conflict of interest.

Electronic supplementary material

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Supplementary Figure 1

Average strand balance across the mitochondrial genome (N = 31) for the Ion PGM™ Sequencing 400 Kit (A) and for the Ion PGM™ Hi-Q™ Sequencing Kit (B) (XLSM 31158 kb).

Supplementary Table 1

Nucleotide positions where the percentage of reads attributed to noise was at or above five percent for either sequencing kit.

Supplementary Table 2

Homology between primer, reference sequence, and read attributed to noise.

Supplementary Table 3

Sequence of the multiple types of noise found between nucleotide positions 13,983 and 13,987.

Supplementary Table 4

Nucleotide positions across entire mitochondrial genome with false deletions.

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Churchill, J.D., King, J.L., Chakraborty, R. et al. Effects of the Ion PGM™ Hi-Q™ sequencing chemistry on sequence data quality. Int J Legal Med 130, 1169–1180 (2016). https://doi.org/10.1007/s00414-016-1355-y

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  • DOI: https://doi.org/10.1007/s00414-016-1355-y

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