Paper

IONIZING RADIATION ALTERS THE TRANSITION/TRANSVERSION RATIO IN

THE EXOME OF HUMAN GINGIVA FIBROBLASTS

Neetika Nath,1,2✝ Lisa Hagenau,1✝ Stefan Weiss,1 Ana Tzvetkova,1,2 Lars R. Jensen,1 Lars Kaderali,2
Matthias Port,3 Harry Scherthan,3 and Andreas W. Kuss1

thus might imply that mismatch repair (MMR) plays a role in the
Abstract—Little is known about the mutational impact of ionizing cellular damage response to IR-induced DNA lesions.
Downloaded from http://journals.lww.com/health-physics by BhDMf5ePHKbH4TTImqenVCkVvhxkEdN1LnrOfMjjv/sTvjKWu9RfKjRt1l2oKBEE on 06/23/2020

radiation (IR) exposure on a genome-wide level in mammalian tis-
sues. Recent advancements in sequencing technology have pro-
vided powerful tools to perform exome-wide analyses of genetic
variation. This also opened up new avenues for studying and char-
acterizing global genomic IR-induced effects. However, genotypes
generated by next generation sequencing (NGS) studies can con-
tain errors, which may significantly impact the power to detect
signals in common and rare variant analyses. These genotyping
errors are not explicitly detected by the standard Genotype Anal-
ysis ToolKit (GATK) and Variant Quality Score Recalibration
(VQSR) tool and thus remain a potential source of false-positive
variants in whole exome sequencing (WES) datasets. In this con-
text, the transition-transversion ratio (Ti/Tv) is commonly used
as an additional quality check. In case of IR experiments, this is
problematic when Ti/Tv itself might be influenced by IR treat-
ment. It was the aim of this study to determine a suitable threshold
for variant filters for NGS datasets from irradiated cells in order
to achieve high data quality using Ti/Tv, while at the same time be-
ing able to investigate radiation-specific effects on the Ti/Tv ratio for
different radiation doses. By testing a variety of filter settings and
comparing the obtained results with publicly available datasets,
we observe that a coverage filter setting of depth (DP) 3 and geno-
type quality (GQ) 20 is sufficient for high quality single nucleotide
variants (SNVs) calling in an analysis combining GATK and
VSQR and that Ti/Tv values are a consistent and useful indicator
for data quality assessment for all tested NGS platforms. Further-
more, we report a reduction in Ti/Tv in IR-induced mutations in
primary human gingiva fibroblasts (HGFs), which points to an ele-
vated proportion of transversions among IR-induced SNVs and
1
Department of Functional Genomics, Interfaculty Institute for Genetics
and Functional Genomics, University Medicine Greifswald, Greifswald,
Germany 2Institute of Bioinformatics, University Medicine Greifswald,
Greifswald, Germany, 3Bundeswehr Institute for Radiobiology, University
of Ulm, München, Germany.
✝Equal contribution
The authors declare no conflicts of interest.
For correspondence contact: Andreas W. Kuss, Universitätsmedizin
Greifswald, Interfakultäres Institut für Genetik und Funktionelle
Genomforschung, Felix-Hausdorff-Str. 8, 17475, Greifswald
kussa@uni-greifswald.de; phone: +49-3834-420-5814; fax: +49-3834-
420-5809
(Manuscript accepted 15 November 2019)
Supplemental digital content is available in the HTML and PDF
versions of this article on the journal’s website www.health-physics.com.
0017-9078/20/0
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DOI: 10.1097/HP.0000000000001251
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Health Phys. 119(1):109–117; 2020
Key words: analysis, statistical; dose; exposure, radiation; genetic
effects
INTRODUCTION
IT IS well known that ionizing radiation (IR) causes lesions in
the DNA; e.g., single strand breaks (SSB) and double strand
breaks (DSB) (Lomax et al. 2013; Sutherland et al. 2000).
Cells possess a number of mechanisms with which DNA
damage can be repaired; however, many of these mecha-
nisms are error-prone (Rodgers and McVey 2016). Mutations
caused by cellular repair mechanisms range from large-scale
(chromosomal rearrangements, large deletions, copy number
variations) to small-scale (short insertions/deletions and point
mutations) (Lomax et al. 2013). Point mutations, where a sin-
gle base in the DNA sequence is substituted by another, can
be categorized into two different types: transitions refer to a
pyrimidine-pyrimidine (C↔T) or purine-purine substitution
(A↔G), whereas transversions refer to pyrimidine-purine
substitutions or vice versa. The transition/transversion ratio
(Ti/Tv) of spontaneous occurring mutations in the whole
human genome is shown to be around 2.1 (DePristo et al.
2011); however, depending on the region of the genome un-
der investigation, a varying range of Ti/Tv ratios have been
observed (Wang et al. 2015). One of the most common tran-
sition mutations is the deamination of methylated cytosine
to thymine. Methylated cytosine occurs in high concentra-
tions in CpG islands, which are commonly located in exonic
regions. Accordingly, with a value of around 3 (Bainbridge
et al. 2011; Guo et al. 2012), the Ti/Tv ratio in the human
exome is higher than in the genome. Previous studies show
a varying range of Ti/Tv ratios in the human genome that is
dependent on the region of the genome under investigation
(Wang et al. 2015). Even though transversions and transi-
tions, and particularly changes in CpG islands, may have
109

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110 Health Physics
epigenetic consequences, the influence of radiation exposure
on the Ti/Tv ratio in healthy tissues has rarely been inves-
tigated. An early study observed a high proportion of
transversions in a mouse cell line containing a mutation re-
porter gene after x irradiation with 10–40 Gy (Yuan et al.
1995). In radiation-associated second malignancies, a rela-
tively low number of mutations was found; however, the ef-
fect on the Ti/Tv ratio was not reported (Behjati et al. 2016).
This lack of information might be owing to the fact that
during single nucleotide variant (SNV) discovery with mod-
ern high throughput sequencing technologies, the Ti/Tv ra-
tio is commonly used as a quality check (Wang et al. 2015),
where a lower-than-expected ratio is considered to indicate
that the SNV set under investigation contains false positive
calls (DePristo et al. 2011). This complicates matters when
Ti/Tv itself might be influenced by the experiment, as could
be the case in studies that aim to detect mutations induced
by IR. Therefore, it was the aim of this study to investigate
the possibility to distinguish IR-specific effects on the Ti/Tv
ratio in primary human gingiva fibroblasts (HGFs).
A popular method for SNP discovery is the Genotype
Analysis ToolKit (GATK) best practices workflow (DePristo
et al. 2011). It contains a standard variant quality filter
(VQSR) that removes low quality variants from the dataset
but does not take genotype quality (GQ) into account. GQ
is a Phred-scaled value representing the degree of confi-
dence that the called genotype is the true genotype, with
higher values reflecting more accurate genotype calls.
Depth (DP) values represent the number of reads passing
the quality control used to calculate the genotype at a spe-
cific site in a specific sample. Higher DP values generally
lead to more accurate genotype calls. Filtering steps based
on these metrics improve the overall variant calling quality
(Carson et al. 2014).
Therefore, in order to achieve high whole exome se-
quencing (WES) data quality using Ti/Tv, while at the same
time being able to investigate radiation-specific effects on
the Ti/Tv ratio for different radiation doses, a filtering strat-
egy was developed: in addition to VQSR, this strategy also
takes GQ and DP into account using a coverage filter based
on these genotype-level quality metrics and the Ti/Tv ratio
as additional quality control to obtain a high quality variant
set. To determine the best parameters for DP and GQ em-
pirically, the control dataset was used; i.e., only those
cells that had not been exposed to radiation. The parame-
ters determined by this procedure were then applied to the
whole dataset.
After that, a sample filter removing SNVs present in
the control dataset (for further details see Nath et al. 2018)
was employed to distinguish radiation-specific Ti/Tv changes
in samples exposed to varying doses of radiation.
To the best of our knowledge, this is the first study in-
vestigating changes in the Ti/Tv ratio of irradiated HGFs.
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METHODS
Cell culture
Primary HGFs were obtained from Provitro AG (Berlin,
Germany) and cultured and treated as previously described in
Weissmann et al. (2016). Cells were irradiated with 240 kV X
rays at 13 mA (YXLON Maxishot, Hamburg, Germany)
filtered with 3 mm beryllium at a dose rate of 1 Gy min−1
as described earlier (Weissmann et al. 2016).
Study design and DNA-sequencing
After exposure of the cells to different doses of X radi-
ation (0, 0.5, 2, and 10 Gy), they were cultured along with
non-irradiated control cells for 16 h (repair interval). DNA
was extracted and purified using the NucleoSpin Tissue
Kit by Macherey Nagel (Düren, Germany) according to
the manufacturers’ instructions and subjected to WES using
three different NGS platforms each time according to the in-
structions provided by the manufacturer of the respective se-
quencing system. Fig. 1 illustrates our study design. Table 1
summarizes key information for the obtained WES datasets,
including sequencing platform, average read length, bioinfor-
matics tools used for mapping and variant calling, as well as
average depth (including standard deviation) for each sample
after variant calling.
Reference sample
To compute the reference Ti/Tv ratio from variants from
human genome and exome samples, variant data from the
third phase of the 1000 Genomes Projects were downloaded
(Auton et al. 2015). In the third phase of the 1000 Genome
Projects, genotypes have been determined by use of whole-
genome sequencing and WES in parallel. A subpopulation
of 49 Utah residents with Northern and Western European
ancestry (CEU) was selected to investigate the Ti/Tv ratio.
We used VCFtools for selecting the genotypes from the ref-
erence dataset (Danecek et al. 2011). Sample IDs are listed
in the supplementary material (please see Supplemental Dig-
ital Content 1, http://links.lww.com/HP/A183). We excluded
the genetic variants lying outside the defined exome target to
calculate the exome specific Ti/Tv ratio. The exome coordi-
nates were downloaded from ftp://ftp.1000genomes.ebi.ac.
uk/vol1/ftp/technical/reference/exome_pull_down_targets/.
Bioinformatics workflow
First, trimmomatic was used to remove adapters from
the reads and to distinguish low-quality reads in the dataset
(Bolger et al. 2014). Second, TopHat (for NextSeq and S5
datasets) or Lifescope (for SOLiD dataset) was used to map
reads to the human genome reference sequence (hg19).
Third, the mapped exome datasets were preprocessed using
packages from the GATK and Picard (McKenna et al. 2010;
Broad Institute 2018). Reads originating from a single DNA
fragment were marked as duplicates using the MarkDuplicate
tool. The reads were then assigned to read groups and to
 Ionizing radiation alters the Ti/Tv ratio c N. NATH ET AL. 111

Fig. 1. Study workflow: HGFs were exposed to different x-ray doses (0.5, 2, and 10 Gy), and subsequently cultured for 16 h (repair interval) before
whole exome sequencing was performed and subsequent data analysis carried out. For comparison, publicly available datasets were used (“Ref-
erence data”) and literature review was performed.

sample identifiers using AddOrReplaceReadGroups. After-
ward, reads were locally realigned using RealignerTargetCreator
and IndelRealigner in order to minimize the number of
mismatching bases that could have appeared due to the
presence of insertions and deletions (InDels) in the dataset.
Subsequent quality scores were optimized with VQSR, which
applies machine learning to model errors empirically and ad-
justs the quality scores accordingly. Lastly, HaplotypeCaller
was used for variant calling, including SNVs and InDels.
For further analysis, only SNVs with heterozygous (0/1) and
homozygous (1/1) genotype were considered.
Coverage filter. Variants were further filtered based on
the quality metrics DP and GQ. The absolute minimum re-
quirement to call a heterozygous variant is three reads, two
of which show the same non-reference base (in order to ex-
clude sequence artefacts affecting only one read); hence, the
lowest value for DP was set at 3. All datasets were analyzed

Table 1. Information related to the exome dataset used in this study.

Sequencing platform SOLiD 5500 xl NextSeq r1 NextSeq r2 Ion S5 XL

(Illumina, San (Illumina) (Thermo Fisher
Diego, CA) Waltham, MA)
Bioinformatics workflow Lifescope/Cashaw -> Tophat -> Tophat -> Tophat ->
(mapping and HaplotyperCaller HaplotypeCaller HaplotypeCaller HaplotypeCaller
variant calling)
Average ReadLength 75 bp forward reads 150 bp 150 bp 200 bp
and 35 reverse read
Sequencing read type Paired end Paired end Paired end Single end
Radiation dose in Gy
(16 h repair interval)
DP average (±stdev)
0 Gy 35.3 (± 37.5) 14.3 (± 25.3) 34.2 (± 38.5) 3.3 (± 1.7)
0.5 Gy 47.5 (± 43.7) 16.3 (± 28.3) 39.6 (± 46.2) 3.9 (± 2.3)
2 Gy 44.7 (± 43.8) 16.1 (± 26.7) 47.8 (± 66.8) 2.7 (± 1.2)
10 Gy 71.1 (± 56.1) 17.9 (± 30.1) 39.6 (± 49.1) 3.3 (± 1.7)

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112 Health Physics
using coverage/quality filter settings with DP values rang-
ing from 3 to 20 in combination with GQ values of 10, 13,
and 20.
Determination of Ti/Tv ratio. Ti/Tv ratios are only an
approximate measure of quality so that a certain range of Ti/Tv
ratios are associated with lower false positives; for example,
the Ti/Tv ratios in high quality exome variant datasets range
between 2.40 and 3.23 (see Table 2 for reference). Ti/Tv ra-
tios for on-target variants were computed using the follow-
ing formula:
count ðchanges ðA↔G jC↔T ÞÞ
Ti=Tv ¼ : ð1Þ
countðchanges ðA↔T =CjG↔T =C ÞÞ
Sample filter. All variants that were present in the non-
irradiated control samples were removed from the irradiated
samples. Variants that were identified in the non-irradiated
controls were assumed not to be IR-induced (Nath et al. 2018).
RESULTS
Literature review for Ti/Tv ratios
First, a review of the pertinent literature was performed,
which included a wide range of information on Ti/Tv from
2011 to 2018. The reported Ti/Tv ratios for exome data
range between 2.4 and 3.23 (Table 2). Some of the studies
included Ti/Tv ratios for whole genome datasets as well,
where these values ranged from 2.00 and 2.15. This is in
concordance with previous observations that the Ti/Tv ratio
is strongly influenced by the selected genome target region.
While only two of the studies used a sequencing platform
other than Illumina, the reported Ti/Tv ratios for both ge-
nome and exome are similar to the values reported by the
Illumina-based studies. This suggests that the Ti/Tv ratio
is mostly platform-independent. Therefore, the Ti/Tv ratio
can be considered to be a stable indicator for quality assess-
ment in SNV datasets.
Distribution of Ti/Tv in public reference datasets
In addition to our literature review (Table 1), 49 exome
datasets from phase three of the 1000 Genome Project
(Auton et al. 2015) were downloaded and analyzed, reveal-
ing Ti/Tv ratios between 2.6 and 2.8 (Fig. 2a). This is also
within the range of previously observed Ti/Tv values from
the literature (Table 2).
Ti/Tv based quality assessment in study datasets
In order to empirically determine suitable coverage fil-
ter parameters for the exome sequences of primary HGFs,
the Ti/Tv ratios for non-irradiated control samples (“0 Gy”,
Fig. 2b) were calculated for a range of DP and GQ values.
Based on minimum requirements for heterozygous variant
calling and overall read coverage, DP values from 3 to 20
and GQ values of 10, 13 and 20 were selected. Fig. 2b dis-
plays the distribution of Ti/Tv ratios for all filter settings in
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July 2020, Volume 119, Number 1
the control datasets. Over all filtering settings, the mean
Ti/Tv values of the control exome datasets ranged between
2.32 (±0.02) and 2.42 (±0.06) for the NextSeq and SOLiD
platforms, while the low coverage dataset (S5) showed a
mean Ti/Tv ratio of 1.39 (±0.7) (Fig. 2b). The observation
pertaining to the low coverage dataset is due to the decreasing
numbers of variants that remain at filter settings with increas-
ing stringency (compare Fig. 3).
The Ti/Tv ratios of variants with a low stringency filter-
ing setting (DP 3, GQ 20) yielded values from 2.2 to 2.6 and
were thus comparable to literature findings (Table 2). For
data from the NextSeq and S5 platforms, an average in-
crease in Ti/Tv ratio by 0.1 and 0.02, respectively, was ob-
served, and thus higher quality variant calls. The SOLiD
dataset was not affected by low stringency filtering and
did not show any changes in Ti/Tv values.
Fig. 3 shows the Ti/Tv ratios and numbers of variants
for different coverage filter settings for all datasets. The dif-
ferences between irradiated and control samples were mini-
mal, indicating that the coverage filter was suitable to use on
datasets from IR experiments.
Using the Ti/Tv ratio as a quality measure, we found
that our filter based on GQ and DP metrics improved the
overall quality of our variant call dataset. A coverage filter
with DP 3 and GQ 20 was sufficient to obtain high quality
variant calls, allowing the inclusion of low coverage datasets
in the analysis in principle.
Suitability of quality filtering for data from different
sequencing platforms
While it could be shown that the filtering procedure
can be used for data from all three sequencing platforms,
platform-specific differences were nevertheless visible (Figs. 2
and 3). SOLiD data showed the least variability in response
to changes in filtering conditions with regard to Ti/Tv values
as well as to the number of variants (Fig. 2). This points to
a particularly low number of false positive calls in these
datasets. For NextSeq data, the Ti/Tv ratio increased after
applying a low stringency filter to the unfiltered dataset
(Fig. 3), but any stricter filtering did not change the Ti/
Tv ratio further. The number of variants, however, decreased
slightly. In contrast, the low coverage dataset (S5 dataset) un-
surprisingly showed more pronounced changes in the Ti/Tv
ratio when stricter filtering conditions were applied. This
was due to the small number of variants with high coverage
and thus rapidly decreasing number of overall variants con-
tributing to the Ti/Tv calculation with increasing coverage fil-
ter stringency.
Distribution of Ti/Tv for before and after
sample filtering
In order to study whether IR has an influence on the
Ti/Tv ratio, coverage filtering with the optimized settings
of DP 3 and GQ 20 was performed. The genotypes
 Table 2. Literature review of reported Ti/Tv values from various studies published between 2011 and 2018, including information on sequencing platforms, variant calling, and filtering prac-
tices as well as sample providence is reported.
Ti/Tv ratio
Whole
Publication Genome Exome SNP calling SNP filtering Data source Sequencing platform Samples Population

Gudbjartsson et al. 2015 2.14 2.41 GATK 2.3.9 GATK best practices, other, sequenced for study Illumina 2636 Icelandair
simple tandem repeats
Tennessen et al. 2012 n.a. 2.93 glfMultiples read depth, coverage, sequenced for study Illumina 2240 European (n = 1351),
quality and others African (n = 1088)
Stubbs et al. 2012 2.07 2.99 Complete Genomics Complete Genomics sequenced for Complete Genomics 31 European
(proprietary software) study/database
Gioia et al. 2018 2.10 n.a. GATK haplotype caller GATK variant quality score sequenced for study Ilumina 1 European
recalibration (VQSR) scheme
Wang et al. 2015 2.06 2.81 GATK Unified Genotyper GATK best practices 1000 Genomes Phase 1 Illumina, SOLiD 1092 Mixed
GATK quality score recalibration,
Bainbridge et al. 2011 2.00 3.00 GATK minimum quality score, sequenced for study SOLiD, Illumina 5 Hispanic, European

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coverage, mapping quality
Zook et al. 2014 2.04 2.60 GATK UnifiedGenotyper VQSR, manual curation of 1000 Genomes, Illumina 1 European (HapMap
and HaplotypeCaller v2.6 systematic sequencing errors Broad (subset) NA12878)
DePristo et al. 2011 2.15 (2.05) 3.27 (2.57) GATK GATK best practices (article 1000 Genomes Illumina 1 European (HapMap
establishes workflow) NA12878)
Ionizing radiation alters the Ti/Tv ratio c N. NATH ET AL.

Tang et al. 2016 n.a. 2.48 to 2.56 GATK 2.8 GATK best practices, VQSR sequenced for study Illumina 72 Australian-aboriginal
Guo et al. 2012 n.a. 2.81 GATK Unified Genotyper genotype quality (GQ) and depth, sequenced for study Illumina 22 Asian
n.a. 2.30 GATK Unified Genotyper using Ti/Tv ratio close to sequenced for study Illumina 6 Asian
expected values
n.a. 3.23 GATK Unified Genotyper 1000 Genomes (Pilot 3) Illumina 6 Caucasian

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113
114 Health Physics July 2020, Volume 119, Number 1

Fig. 2. (a) Boxplot showing the Ti/Tv ratios in 49 public exome datasets from the 1000-Genome Project (Auton et al. 2015) central European pop-
ulation sample (box plot) and the unfiltered Ti/Tv values of non-irradiated samples from this study (symbols as indicated to the right of the plot); (b)
boxplot representing the distribution of TiTv ratios that were obtained after different filter cutoffs for depth (DP) and genotype quality (GQ), on
y-axis. On x-axis different sequencing platforms (NextSeq, SOLiD, ION S5) used for generating WES data for non-IR samples.

identified in control samples were then subtracted from the Interestingly, there was no consistent irradiation dose
datasets of irradiated samples in the sample-filtering step dependence of SNV counts. However, a reduction in
(Nath et al. 2018). This led to a removal of approximately Ti/Tv ratio from 2.2–2.5 to 1.0–1.9 was observed for all
80% of SNVs from all exome datasets (Table 3). samples and for all sequencing platforms (Fig. 4), which

Fig. 3. Heatmap representing the Ti/Tv ratios for different depth (DP) and genotype quality (GQ) filtering cutoffs in exome sequencing datasets
from three different sequencing platforms (facets) after treatment with different doses of IR and a subsequent repair interval of 16 h. Heatmap col-
oring indicates Ti/Tv ratios of 2–2.5, variant numbers after filtering are represented by the size of the circle.
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 Ionizing radiation alters the Ti/Tv ratio c N. NATH ET AL. 115

Table 3. SNV counts before and after sample filtering in exome data sets used in this study.

Sequencing machine SOLiD 5500 xl NextSeq r1 NextSeq r2 Ion S5 System

Genetic Analyzer (Illumina) (Illumina) (Thermo Fisher)
Bioinformatics workflow Lifescope -> Tophat -> Tophat -> Tophat ->
(mapping and HaplotyperCaller HaplotypeCaller HaplotypeCaller HaplotypeCaller
variant calling)
Radiation dose in Gy
(16 h repair interval)
SNV counts
before / after 0.5 Gy 28,032 / 1,487 154,516 / 38,838 115,288 / 14,068 337,693 / 9,940
sample filter
2 Gy 27,601 / 1,260 144,836 / 30,355 143,110 / 37,499 21,091 / 2,731
10 Gy 28,996 / 2,499 157,003 / 38,966 120,231 / 20,685 29,786 / 5,836

indicates an increase in the number of transversions
among the IR-induced SNVs.
DISCUSSION
In this study, we analyzed the IR-induced mutational
spectrum of IR-exposed HGFs by WES. We show that a
combination of a DP value of 3 and a GQ value of 20 after
Fig. 4. Ti/Tv ratio in for non-IR and IR-exposed samples before and
after sample filtering for three different sequencing platforms. After
sample filtering, DP=3 and GQ=20 were selected as a coverage filter
parameters.
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GATK HaplotypeCaller variant calling is sufficient for
quality filtering of high throughput sequencing data for
the analysis of IR-induced sequence alterations. The results
were platform-independent and allowed the inclusion of a
low coverage dataset in the analysis.
Comparison of the Ti/Tv ratios for exome data from
this study with previous findings (Table 2) and the results
obtained from publicly available datasets (Fig. 2a) showed
that our dataset yielded similar results (Fig. 3). The large
range for Ti/Tv values in the previous studies involving ex-
ome data can partly be explained by the different parameter
settings used for the respective bioinformatics workflows,
the varying filtering procedures applied, and/or the nature
of the investigated variants. Some studies included values
for previously unobserved and thus unconfirmed variants,
which could have resulted in lower Ti/Tv ratios as compared
to datasets comprising mostly or exclusively known variants.
In such cases, the authors assumed that this effect was the
result of sequencing or variant calling errors among the pre-
viously unconfirmed variants. Other variables include the
regions for which the Ti/Tv ratio was calculated (e.g., exonic
or consensus coding sequence), the data source and the num-
ber of samples included in the respective study.
Nonetheless, our findings for untreated as well as for
irradiated HGFs prior to sample filtering (i.e., the removal
of SNVs that are already present in untreated cells) agree
with these previous results.
Due to the high sequencing accuracy of the SOLiD
platform, datasets generated with the SOLiD sequencer were
already at a high quality level so that coverage filtering had
little impact on data quality, whereas data from the NextSeq
sequencer were improved. Filtering the data from the S5
sequencer produced less consistent results, which can be
explained by the low overall coverage of this dataset. It
can be concluded that the choice of sequencing technique
has an influence on the filtering strategy required.
Our WES results showed no consistent correlation be-
tween radiation dose and SNV count. One possible explana-
tion for this might be a DNA repair bias at euchromatic
(transcribed) loci, where DNA repair mechanisms are highly

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116 Health Physics
efficient, while in heterochromatic regions this seems to be
less so. As WES focuses on transcribed areas of the genome,
it misses untranscribed and thus less efficiently repaired
areas with a higher variant load. Hence, the variant counts
observed by our exome-based analysis may not include all
IR-induced changes.
After removal of all SNVs present in untreated cells, a
reduction in Ti/Tv ratios was observed. This result was
platform-independent and could be reproduced in all inves-
tigated biological replicates. At this point, however, the
lower coverage dataset (S5) was less conclusive than those
from the other two platforms, showing that for this kind of
analysis a higher coverage should be aimed for, if possible.
The lower Ti/Tv ratio for IR-induced SNVs may in part be
explained by a relatively small proportion of transitions aris-
ing from spontaneous deamination of 5-methylcytosine resi-
dues. These are at least partially responsible for the normally
observed surplus of transitions, and thus higher Ti/Tv values,
in unirradiated cells. Our experiments were carried out with
a 16-h repair interval, so that the cells had little time to ac-
cumulate transitions of this kind. Still, in all cases the results
clearly demonstrate that IR leads to a relative increase in the
number of transversions among the observed IR-induced
SNVs. This corresponds with recent studies on plant ge-
nomes and transcriptomes where, after neutron irradiation,
a lower Ti/Tv ratio was observed in IR-induced mutations
as compared to spontaneous mutations (Zhou et al. 2019;
Li et al. 2016; Shirasawa et al. 2019).
Our finding is also in agreement with the earlier findings
of Yuan et al. (1995) in murine cells and could point to a more
prominent contribution of the mismatch repair (MMR) system
to the IR-induced DNA-damage response in mammalian cells
than previously assumed: MMR strongly favors the repair of
transitions over transversions (Lujan et al. 2012), which could
account for an increase in the number of transversions. Still,
very little is known so far about a possible involvement of
the MMR system in the context of IR-induced DNA damage
(Martin et al. 2010). In this respect, it is of note that the upreg-
ulation of the MMR system in inhabitants of the high natural
background radiation area of Kerala was published while this
paper was in press (Bakhtiari et al. 2019). (https://www.ncbi.
nlm.nih.gov/pubmed/31323602). Therefore, additional stud-
ies are required to further elucidate this question.
CONCLUSION
Taken together, our results provide strong evidence that
a coverage filter setting of DP 3 and GQ 20 is sufficient for
high quality SNV calling in datasets from IR experiments
and that Ti/Tv ratios are a consistent and useful indicator
for data quality assessment for all tested NGS platforms. Fur-
thermore, we report a drop in Ti/Tv ratios in IR-induced mu-
tations in HGFs, which points to an elevated proportion of
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July 2020, Volume 119, Number 1
transversions among IR-induced SNVs and thus might imply
that MMR plays a role in the cellular damage response to
IR-induced DNA lesions.
Acknowledgments—We thank Jessica Müller, Christian Sperling, and Corinna
Jensen for excellent technical help. AWK received funding for this project
through the German Federal Ministry of Defense (E/U2AD/CF520/DF554).
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