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Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors

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Abstract

Human remains can be severely affected by the environment, and the DNA may be damaged, degraded, and/or inhibited. In this study, a DNA sample (at 1 ng DNA target input in triplicate) was spiked with five concentrations of five inhibitors (humic acid, melanin, hematin, collagen, and calcium) and sequenced with both the HID-Ion AmpliSeq™ Library Kit and ID panel on the Ion PGM™ System and the ForenSeq DNA Signature Prep Kit on the MiSeq FGx™. The objective of this study was to compare the baseline tolerance of the two sequencing chemistries and platforms to common inhibitors encountered in human remains recovered from missing person cases. The two chemistries generally were comparable but not always susceptible to the same inhibitors or at the same capacity. The HID-Ion AmpliSeq™ Library Kit and ID panel and the ForenSeq DNA Signature Prep Kit both were susceptible to humic acid, melanin, and collagen; however, the ForenSeq kit showed greater inhibition to melanin and collagen than the AmpliSeq™ kit. In contrast, the ForenSeq kit was resistant to the effects of hematin and calcium, whereas the AmpliSeq™ kit was highly inhibited by hematin. Short tandem repeats (STRs) and single nucleotide polymorphisms (SNPs) showed the same trend among inhibitors when using the ForenSeq kit. Generally, locus read depth, heterozygote allele balance, and the numbers of alleles typed were inversely correlated with increasing inhibitor concentration. The larger STR loci were affected more so by the presence of inhibitors compared to smaller STR amplicons and SNP loci. Additionally, it does not appear that sequence noise is affected by the inhibitors. The noise percentage, however, does increase as the inhibitor concentration increases, due to the decrease in locus read depth and not likely because of chemistry effects.

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Acknowledgements

The authors would like to thank the individuals who provided the biological sample used in this study.

Funding

This project was supported in full by the National Institute of Justice (NIJ) Award Number 2015-DN-BX-K066 awarded to Sam Houston State University (SHSU) and the University of North Texas Health Science Center (UNTHSC).

Author information

Author notes

  1. Kyleen Elwick and Xiangpei Zeng are contributed equally to this work

Authors and Affiliations

  1. Sam Houston State University, Huntsville, TX, USA

    Kyleen Elwick & Sheree Hughes-Stamm

  2. Center for Human Identification, University of North Texas Health Science Center, 3500 Camp Bowie Blvd., Fort Worth, TX, 76107, USA

    Xiangpei Zeng, Jonathan King & Bruce Budowle

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

    Bruce Budowle

Authors
  1. Kyleen Elwick
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  2. Xiangpei Zeng
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Corresponding author

Correspondence to Xiangpei Zeng.

Electronic supplementary material

Supplemental Table 1

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Supplemental Fig. 1

The average SNP loci read depth (Ion AmpliSeq™ Library Kit and ID panel) of a 1 ng DNA sample spiked with five concentrations of five PCR inhibitors. Concentration 0 means no inhibitor added. The inhibitor concentrations are listed in Table 1. Data presented as average ± standard deviation (N = 3) (XLSX 12 kb)

Supplemental Fig. 2

The average STR loci read depth (ForenSeq™ DNA Signature Prep Kit) of a 1 ng DNA sample spiked with five concentrations of five PCR inhibitors. Concentration 0 means no inhibitor added. The inhibitor concentrations were listed in Table 1. Data presented as average ± standard deviation (N = 3) (XLSX 15 kb)

Supplemental Fig. 3

The average SNP loci read depth (ForenSeq™ DNA Signature Prep Kit) of a 1 ng DNA sample spiked with five concentrations of five PCR inhibitors. Concentration 0 means no inhibitor added. The inhibitor concentrations were listed in Table 1. Data presented as average ± standard deviation (N = 3) (XLSX 15 kb)

Supplemental Fig. 4

The heatmap of alleles typed at each SNP locus with five concentrations of melanin (Ion AmpliSeq™ Library Kit and ID panel) (TIFF 60 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 5

The heatmap of alleles typed at each STR locus with five concentrations of melanin (ForenSeq™ DNA Signature Prep Kit) (TIFF 48 kb)

High resolution (GIF 6 kb)

Supplemental Fig. 6

The heatmap of alleles typed at each SNP locus with five concentrations of melanin (ForenSeq™ DNA Signature Prep Kit) (TIFF 55 kb)

High resolution (GIF 6 kb)

Supplemental Fig. 7

The heatmap of alleles typed at each SNP locus with five concentrations of hematin (Ion AmpliSeq™ Library Kit and ID panel) (TIFF 57 kb)

High resolution (GIF 6 kb)

Supplemental Fig. 8

The heatmap of alleles typed at each STR locus with five concentrations of hematin (ForenSeq™ DNA Signature Prep Kit) (TIFF 35 kb)

High resolution (GIF 2 kb)

Supplemental Fig. 9

The heatmap of alleles typed at each SNP locus with five concentrations of hematin (ForenSeq™ DNA Signature Prep Kit) (TIFF 40 kb)

High resolution (GIF 2 kb)

Supplemental Fig. 10

The heatmap of alleles typed at each SNP locus with five concentrations of collagen (Ion AmpliSeq™ Library Kit and ID panel) (TIFF 54 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 11

The heatmap of alleles typed at each STR locus with five concentrations of collagen (ForenSeq™ DNA Signature Prep Kit) (TIFF 42 kb)

High resolution (GIF 3 kb)

Supplemental Fig. 12

The heatmap of alleles typed at each SNP locus with five concentrations of collagen (ForenSeq™ DNA Signature Prep Kit) (TIFF 54 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 13

The heatmap of alleles typed at each SNP locus with five concentrations of calcium (Ion AmpliSeq™ Library Kit and ID panel) (TIFF 55 kb)

High resolution (GIF 4 kb)

Supplemental Fig. 14

The heatmap of alleles typed at each STR locus with five concentrations of calcium (ForenSeq™ DNA Signature Prep Kit) (TIFF 36 kb)

High resolution (GIF 2 kb)

Supplemental Fig. 15

The heatmap of alleles typed at each SNP locus with five concentrations of calcium (ForenSeq™ DNA Signature Prep Kit) (TIFF 41 kb)

High resolution (GIF 2 kb)

Supplemental Fig. 16

The noise percentages of SNPs (Ion AmpliSeq™ Library Kit and ID panel) of reference samples and DNA samples spiked with five concentrations of melanin. X axis is locus read depth, Y axis is noise percentage (TIFF 153 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 17

The noise percentages of SNPs (Ion AmpliSeq™ Library Kit and ID panel) of reference samples and DNA samples spiked with five concentrations of hematin. X axis is locus read depth, Y axis is noise percentage (TIFF 132 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 18

The noise percentages of SNPs (Ion AmpliSeq™ Library Kit and ID panel) of reference samples and DNA samples spiked with five concentrations of collagen. X axis is locus read depth, Y axis is noise percentage (TIFF 154 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 19

The noise percentages of SNPs (Ion AmpliSeq™ Library Kit and ID panel) of reference samples and DNA samples spiked with five concentrations of calcium. X axis is locus read depth, Y axis is noise percentage (TIFF 153 kb)

High resolution (GIF 4 kb)

Supplemental Fig. 20

The noise percentages of STRs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of humic acid. X axis is locus read depth, Y axis is noise percentage (TIFF 148 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 21

The noise percentages of STRs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of melanin. X axis is locus read depth, Y axis is noise percentage (TIFF 134 kb)

High resolution (GIF 4 kb)

Supplemental Fig. 22

The noise percentages of STRs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of hematin. X axis is locus read depth, Y axis is noise percentage (TIFF 159 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 23

The noise percentages of STRs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of collagen. X axis is locus read depth, Y axis is noise percentage (TIFF 170 kb)

High resolution (GIF 6 kb)

Supplemental Fig. 24

The noise percentages of STRs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of calcium. X axis is locus read depth, Y axis is noise percentage (TIFF 155 kb)

High resolution (GIF 6 kb)

Supplemental Fig. 25

The noise percentages of SNPs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of humic acid. X axis is locus read depth, Y axis is noise percentage (TIFF 155 kb)

High resolution (GIF 4 kb)

Supplemental Fig. 26

The noise percentages of SNPs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of melanin. X axis is locus read depth, Y axis is noise percentage (TIFF 136 kb)

High resolution (GIF 4 kb)

Supplemental Fig. 27

The noise percentages of SNPs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of hematin. X axis is locus read depth, Y axis is noise percentage (TIFF 146 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 28

The noise percentages of SNPs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of collagen. X axis is locus read depth, Y axis is noise percentage (TIFF 136 kb)

High resolution (GIF 5 kb)

Supplemental Fig. 29

The noise percentages of SNPs (ForenSeq™ DNA Signature Prep Kit) of reference samples and DNA samples spiked with five concentrations of calcium. X axis is locus read depth, Y axis is noise percentage (TIFF 132 kb)

High resolution (GIF 4 kb)

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Elwick, K., Zeng, X., King, J. et al. Comparative tolerance of two massively parallel sequencing systems to common PCR inhibitors. Int J Legal Med 132, 983–995 (2018). https://doi.org/10.1007/s00414-017-1693-4

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