1. science
2. biology
3. molecular biology

DNA storage under extreme temperature conditions does not affect
performance in HLA genotyping via next-generation sequencing
Shana L McDevitt1
SLM: [email protected]
Michael E Hogan2
MEH:[email protected]
Derek J Pappas1
DJP: [email protected]
Lily Y Wong2
LYW: [email protected]
Janelle A Noble1, §
JAN: [email protected]
1
Children’s Hospital Oakland Research Institute, Oakland, California, USA
2
GenTegra LLC, Pleasanton, California, USA
§
Corresponding author
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Abstract
Background
Stable dry-state storage DNA is desirable not only to minimize required
storage space but also to reduce electrical and shipping costs. DNA purified
from various commercially available dry-state stabilization matrices has been
used successfully in downstream molecular applications, e.g., quantitative
polymerase
chain
reaction
(PCR),
microarrray,
and
sequence-based
genotyping. However, standard DNA storage conditions in most laboratories
still include freezing of liquid DNA. Broad implementation of dry-state, longterm DNA storage requires enhancement of such dry-state DNA stabilization
products to control for temperature fluctuations at specimen collection, transit,
and storage. This study tested the integrity of genomic DNA subjected to longterm storage on GenTegraTM DNA stabilization matrices (GenTegra LLC,
Pleasanton, CA) at extreme conditions, as defined by a four year storage
period at ambient temperature with an initial incubation for seven months at
37°C,
56°C,
or
ambient
temperature.
Subsequently,
purified
DNA
performance and integrity was measured by quantitative PCR and nextgeneration sequencing (NGS) based human leokocyte antigen (HLA)
genotyping.
Results
High molecular weight genomic DNA samples were recovered from the
GenTegraTM product matrix and exhibited integrity comparable to a highly
characterized commercial standard under assessment by qPCR. Samples
were genotyped for classical HLA loci using next generation sequencingbased methodolgy on the Roche 454 GS Junior instrument. Amplification
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efficiency, sequence coverage, and sequence quality were all comparable
with those produced from a cell line DNA sequenced as a control. No
significant differences were observed in the mean, median, or mode quality
scores between samples and controls (p≥0.4).
Conclusions
Next generation HLA genotyping was chosen to test the integrity of
GenTegraTM treated genomic DNA due to the requirment for long sequence
reads to genotype the highly polymorphic classical HLA amplicon genes.,
Results of this experiment demonstrate the efficacy of the GenTegraTM
product as a suitable genomic DNA preservation tool for collection, long-term
biobanking, or shipping of DNA at fluctuating and high temperatures.
-3-
Keywords
Human Leukocyte Antigen, Next Generation Sequencing, Bio-banking, DNA
preservation
Background
The preservation of purified DNA, during long-term biobanking or shipping,
has become a major concern in the field of applied genetics and public health
screening [1]. For cryogenic preservation of high-value DNA samples,
mechanical and liquid nitrogen storage for biobanking, or dry ice and chemical
packs for shipping are proven technologies. However, cryogenic preservation
methods introduce substantial cost and some measure of risk, such as
chemical refrigerant failure during shipping or power loss. Refrigeration-free
biosample preservation could dramatically reduce the cost and risk associated
with cryogenic preservation. Consequently, refrigeration-free approaches to
purified DNA preservation have been developed that preserve purified DNA
via desiccation in the presence of added chemical stabilizers. Successful dry
state DNA preservation at lab ambient temperatures for many months has
been reported, resulting in DNA of sufficient quality to support routine
laboratory analyses like quantitative polymerase chain reaction (qPCR),
sequence based typing (SBT), and microarrays [2-5]. However, one
compelling application of refrigeration-free DNA stabilization is for collection of
samples in the field, where the conditions are likely to be far more harsh than
in the laboratory setting.
Here we assess the performance of GenTegraTM (GenTegra LLC, Pleasanton,
CA) under conditions which emulate “worst-case” ambient temperature
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storage or shipping. Using high-quality DNA purified from freshly collected
blood, DNA was subject to room termpature storage for four years, which
included a 6-month initial treatment at elevated temperatures (37°C or 56°C)
to reflect conditions that could be encountered during sample collection,
shipping, or long-term DNA banking. Integrity of the DNA stored under these
adverse conditions was compared to that of DNA stored under standard
conditions. Performance of the DNA stored under adverse conditions was
tested by its use in 2.5 kb qPCR and in next-generation sequencing (NGS)
based human luekocyte antigen (HLA) genotyping. HLA genotyping is
complicated by extreme HLA sequence polymorphism; 10,533 HLA alleles
have been documented as of January, 2014 (https://www.hla.allelles.org).
Methods
DNA storage and purification
Genomic DNA was purified using a Qiagen MidiPrep spin column (Qiagen
Corp, Hilden, Germany) from two freshly collected venous blood samples
(Memorial Blood Centers, St. Paul, MN). DNA concentration was measured
by both Nanodrop UV/VIS spectrospcopy (Thermo Scientific, Waltham, MA)
and Qubit fluorimetry (Life Technologies, Carlsbad, CA) . A total of 18 aliquots
of DNA, 17 from one blood sample and one from the second blood sample,
were arbitrarily split into two groups (C1-C9 and D1-D9) and stored in three
plates. 3 g (C samples) or 5 g (D samples) of purified DNA sample (in TE
buffer) was applied per well of a microplate containing the GenTegraTM DNA
stabilization matrix (GenTegra LLC, Pleasanton, CA). Each plate contained
three samples from each group. Each DNA sample in GenTegra was then airdried per the manufacturer’s recommendation; the wells were sealed and then
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stored as follows for 4 years. One plate was heated to 37°C in an oven for 7
months, then transferred to a warehouse for ambient temperature storage
(~25°C) for 3 years, 5 months (Figure 1A). A second plate was heated to 56°C
in an oven for 7 months, then transferred to a warehouse for ambient
temperature storage (~25°C) for 3 years, 5 months (Figure 1A). A third plate
was kept in a warehouse at ~25°C for the entire 4 year time period.
Subsequent to the 4 year storage, the 3 g (C group) and 5 g (D group)
samples were rehydrated with 20 L and 33 L water, respectively. 100 ng of
each DNA sample (as assessed by NanoDrop ) was then subjected to native
0.8% agarose gel electrophoresis with ethidium staining (Life Technologies,
Carlsbad, CA) (Figure 1B).
qPCR Assessment of DNA Quality
25 ng of each stored DNA sample was subjected to quantitative real-time
PCR (qPCR) targeting a 2.5 kb region of mitochondrial DNA. Each reaction
also
included
250
nM
of
forward
CGCACGGACTACAACCACGAC-3’),
reverse
primer
mt2568-F
(5’-
primer
mt2568-R
(5’-
CTGTGGGGGGTGTCTTTGGGG-3’), and FastStart SYBR Green with ROX
(Roche Applied Sciences, Indianapolis, IN). qPCR was performed on the
Applied Biosystems 7500 Fast Real-time PCR System (Life Technologies,
Foster City, CA). The thermal cycling conditions were: 1) 95°C for 10 min; 2)
40 cycles of 95°C for 30 sec, 68°C for 1 min, 70°C for 2 min, 3) 77°C for 30
sec, followed by a dissociation step post-cycling. qPCR was performed in
triplicate and the average CT value for each sample was plotted in Figure 1C,
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relative to CTs obtained for 25 ng of a highly validated control DNA (Roche
Applied Sciences, Indianapolis, IN).
HLA genotyping using next-generation sequencing (Roche 454)
Polymerase chain reaction (PCR) products (amplicons), were generated from
purified genomic DNA (gDNA) from all eighteen samples, 6 samples per
condition, stored dry with the GenTegraTM product and from a cell line gDNA
sample as control. Fourteen amplicons were generated from each DNA
sample using commercially available Roche 454 HLA GType Medium
Resolution (MR) and High Resolution (HR) HLA fusion primer assay plates
(Roche 454, Branford, CT) [6]. Roche 454 GType MR assay plates include
fusion primers to amplify HLA-A, -B, and -C exons 2 and 3, HLA-DQB1 exon
2, and HLA-DRBX; where X includes all DRB genes in addition to HLA-DRB1.
Roche 454 GType HR assay plates include fusion primers to amplify HLA-A, B, and -C exons 4, HLA-DQB1 exon 3, HLA-DQA1 exon 2, and HLA-DPB1
exon 2. Roche 454 fusion primers consist of a 10-base multiplex ID (MID) tag,
flanked by a locus-specific primer on the 3’ end, and an “A” or “B” 454-specific
primer adaptor sequence on the 5’ end [6, 7]. The MID tag serves as a
barcode for sample identification by data analysis programs.[7, 8]
PCRs were performed in a 25 μl volume. Each reaction contained 20 ng of
genomic DNA, 2 units of AmpliTaq Gold Polymerase (Life Technologies,
Carlsbad, CA), 1X AmpliTaq PCR Gold Buffer (Life Technologies, Carlsbad,
CA), 1.5 mM AmpliTaq Gold MgCl2 Solution (Life Technologies, Carlsbad,
CA), 0.3 μM of PCR Grade Nucleotide Mix (Roche Applied Sciences,
Indianapolis, IN), and 10% Ameresco brand glycerol (Life Technologies,
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Carlsbad, CA). Genomic DNA and PCR mastermix were aliquoted directly into
the Roche 454 GType primer assay plates and PCR was performed using the
thermal cycler GenAmp PCR 9700 system (Life Technologies, Carlsbad, CA).
Cycling conditions were: 1) 94°C for 5 minutes, 2) 31 cycles of 94°C for
15sec, 62°C for 15s, and 72°C for 30sec, 3) 72°C for 8 minutes.
PCR products were visualized with 2% agarose “e-gel” electrophoresis (Life
Technologies, Carlsbad, CA) and with the Advanced Analytical 96-Capillary
Gel
Electrophoresis
based
Fragment
Analyzer
(Advanced
Analytical
Technical Instruments, Ames, IA). PCR products were pooled, based on
Quant-iT PicoGreen dsDNA reagent concentration data (Life Technologies,
Carlsbad, CA), into a single, eqimolar amplicon library, purified with Agencourt
AMPure XP beads (Beckman Coulter, Pasadena, CA), re-quantified using
Quant-iT PicoGreen dsDNA reagent, and mixed with 454 capture beads
(Roche 454, Branford, CT) after dilution to 1 x 106 molecule per microliter
concentration. Individual HLA amplicon molecules within the library were
clonally amplified and bound to 454 capture beads in emulsion PCR [6,
7].DNA bound beads were enriched and deposited onto a single region 454
Titanium PicoTiter plate (Roche 454, Branford, CT) [6, 7]. Molecules were
pyrosequenced on the Roche 454 GS Junior system (Roche 454, Branford,
CT) [6, 7].
Data analysis and Statistics
FASTA formatted sequencing reads were filtered based on alignment to an
International Immunogenetics Information System (IMGT) [9] HLA reference
-8-
database using the Roche 454 Amplicon Variant Analyzer (Roche 454,
Branford, CT). Sequencing reads from a subsequent FASTA file were
analyzed with SCORETM software and Conexio ATF AssignTM HLA genotyping
softwares. 454 GS Junior data processing tools, SFFfile and SFFinfo, were
used to parse files and to extract sequencing adaptor trimmed and untrimmed
sample-specific base calling quality metric scores using manufacturer
protocols. Raw base quality scores for all sequencing reads, from the original
SFF file, were then analyzed per sample in reference to controls in R (R Core
Team). Wilcoxon rank sum tests were used to test for significance between
sample and control means; significance threshold was set at p ≤ 0.05.
Results and Discussion
Stored genomic DNA maintained integrity
The integrity of eighteen gDNA samples recovered from the GenTegraTM
product matrix was confirmed. As seen in Figure 1B, a limiting, high molecular
weight DNA band, with apparent size greater than 40 kilobases was detected
via native 0.8% agarose electrophoresis for all samples, indicating that, upon
recovery from the GenTegraTM product, DNA chain length remains in the high
molecular weight range expected for genomic DNA samples.
Real-time PCR assay validated mitochondrial DNA integrity
The quality of DNA obtained after long term dry state storage was assessed
by qPCR, targeting a 2.5 kb region of human mitochondrial DNA. The target
size is approximately 10 times longer than that typically employed for qPCR
and, thus, is capable of assessing DNA integrity by PCR in the presence of
lesions at a density as low as 0.5 per kilobase. In this assay, CT values
obtained by qPCR are compared to that obtained from a highly characterized
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commercial human gDNA standard (PN 11691112001; Roche Applied
Sciences, Indianapolis, IN). CT values obtained for the gDNA samples stored
in GenTegra were found to be in the range of 19-20 cycles, independent of
storage condition. CT values for all stored samples were found to be
approximately 3 cycles fewer than the un-stored, control DNA. Since DNA
damage, or the presence of contaminants would increase, rather than
decrease CT values, the data in Figure 1C demonstrate that the DNA samples
stored in the GenTegra matrix are at least as intact (and free from PCR
inhibitors) as the control DNA.
Stored genomic DNA amplifies for troublesome HLA loci
All eighteen samples and the positive control amplified successfully for each
of the target HLA loci. Amplicons were visualized by 2% agarose gel
electrophoresis and by capillary gel electrophoresis. Non-specific amplification
was not observed (Figure 2). At 740 base pairs (bp), HLA-A exon 4 amplicons
are the largest in the set of foureen. Figure 2 displays capillary gel
electrophoresis trace overlays of HLA-A exon 4 amplicons from all 18
samples and postive control PCR products around the target size, 740 bp.
Amplicon concentrations ranged from 10.5 nanograms per microliter (ng/ul) to
17.2 ng/ul with a standard deviation of ± 2.23 ng/ul for the longest HLA
amplicon (HLA-A exon 4) as compared to postive control HLA-A exon 4
amplicon concentrations, 13.2 ng/ul and 13.7 ng/ul ± 0.23 ng/ul. In general,
amplicon concentrations ranged from 5.56 ng/ul to 28.3 ng/ul for the
experimental amplicons, as compared to control amplicon concentration
ranges of 6.64 ng/ul to 24.4 ng/ul. The lowest concentrations in both groups
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were observed for HLA-C exon 3 (673bp), which was the most difficult locus
to amplify, possibly due to inefficient primer annealing.
Stored genomic DNA effictively used to genotype HLA
Three sequencing runs were performed in total. Each run included all HLA
amplicons for six experimental samples and one control. The same control
library was sequenced in each run to control for run-to-run variation. The runs
showed some variability in the number of quality filter passed sequencing
reads, 126,665 reads, 110,766 reads, and 134,317 reads, respectively (454
software manual). All three sequencing runs, however, far exceed the 70,000
quality passed filter read benchmark set by Roche 454 [10].
As expected, identical HLA genotypes were assigned for 17 out of 17
identically sourced samples. The single sample sourced from a different
individual produced a different genotype. Control genotypes were identical for
each of the HLA loci among the three sequencing runs, demonstrating the
consistency of the equencing and genotype calling processes.
111 of 126 total genotypes (18 samples sequenced for 7 genes) were
assigned without the need for manual user editing in the Conexio ATF
AssignTM HLA genotyping software. Minimal user edits to remove sequence
containing obvious sequencing error were necessary to assign genotypes to
11 out of the initial unassigned 15 genotypes. The remaining 4 genotypes
cannot be assigned due to mid HLA-B exon 3 sequencing error or cannot be
assigned without excluding HLA-A exon four data. However, these failed
assignments are attributable to limitations in 454 sequencing chemistry rather
than the initial DNA integrity.
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454 sequencing read length limitations
Roche 454 pyrosequencing chemistry delivers the longest read lengths of all
clonally based sequencing platforms [10, 11], with the ability to generate high
quality reads 400 bp in length on either the 454 GS FLX or 454 GS Junior
platforms [10]. HLA-A exon 4 and HLA-C exon 2 amplicons, 740 and 673 bp
respectively, exceed the size of exons and far exceed the length limitations of
the sequencing chemistry. For both amplicons, the exon lies near the
upstream end; thus, the quality of the exon sequence from the forward
sequence reactions was higher than that from the reverse sequences. In the
reverse sequence reactions, the exon sequence was at the end of the read,
and DNA sequencing tends to decrease in quality with read length [10]. Figure
3 shows that, within this data set, all untrimmed sequencing reads maintain a
high level of base calling quality (Q), where Q0 and Q40 are the minimum and
maximum quality score range, respectively, until read lengths approach 430
bases. At this point, the ability of the sequencing software to call bases
accurately steadily decreases. Moreover, when comparing the quality scores
from reads at nucleotide position 423 (Figure 3d), distribution of quality scores
were not different for the test samples as compared to the control samples
(adjusted p = 1).
HLA-A exon 4 and HLA-C exon 3 reverse reads for all samples in the second
sequencing run show evidence of increased sequencing error at the 3’ end of
reverse reads. Failure to generate reverse direction sequences long enough
to capture full HLA-A exon 4 and full HLA-C exon 3 sequence was also
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observed in the control sample from the second sequencing run. Only HLA-A
genotypes for samples C8 and D3 were left unassigned. Genotypes for these
samples can be determined, albeit with increased ambiguity, upon the
exclusion of all exon 4 sequences.
454 sequencing homopolymer detection limitations
Genotype assignments for the 17 identically sourced samples can be used to
distinguish sequencing error from genomic variation where failed genotype
assignments were made. Five HLA-B genotypes showed evidence of a base
calling discrepancy between the forward and reverse sequence reads from
the same chromosome near the center of exon 2 near a high G-C rich region
in the template strands.
Regardless of input DNA quality, homopolymers (i.e., multiple molecules of
the same nucleotide) can be problematic for 454 chemistry [10]. 454
sequencing data are represented by a series of intensity values, or a
flowgram, for iterative deoxynucleotide flows during which multiple nucleotide
incorporations may occur in the absence of a 3’ chain terminator which can
lead to substitution or deletion error [12]. Nucleotide sequences off-set by one
base in either direction, cannot be aligned with confidence to a HLA
reference.
Three of the five samples with noted sequencing error (C1, C2, and C3) were
in the 56°C storage group while the remaining two samples (D2 and D3) were
from the ambient temperature storage group. No other replicates showed
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evidence of a similar issue with HLA-B sequence. Each of the noted samples
show a deletion in the reverse reads for both alleles at base position 389,
which directly follows a G-C rich homopolymer region. Notably, the forward
reads for these alleles per sample have no evidence of sequencing error.
However, D7, D8, and D9, also from the 56°C worst-case scenario treatment
group, all showed no evidence of sequencing error suggesting that the errors
reflected in samples C7-C9, D2 and D3 are not due to the storage conditions
or the subsequent quality of genomic DNA but are rather attributable to
reduced sequencing run quality. Vandenbroucke et al. suggested that every
amplicon has its own error profile dependent upon its originating sequence
[10, 13]. The standard error for this data set appears high because 17 identical
samples have the same HLA-B genotype (HLA-B*07:02:01, HLA-B*55:01:01),
consisting of two alleles with identical sequence contributing to a high
propensity for this homopolymer error.
Stored sample sequencing quality scores mirrored controls
Quality scores are assigned to each base for each sequencing read by the
454 sequencing software, representing the log-probability that a base was not
an overcall [10, 12, 14]. These scores are reported in a FASTA like format. 454
base quality scores are represented by Phred equivalent quality scores (Q = 10 * log10 (error rate)) ranging from Q0-Q40, where a Q30 score represents a
base-call with a probablility of 1/1000 of being incorrect while a Q40 score
represents a 1/104 chance of being incorrect, in other words, representing a
99.99% accuracy rating (https://www.broadinstitute.org) [10, 15]. No significant
differences were observed (p ≥ 0.4) in the mean, median, or mode quality
scores between samples and control (Figure 4). Quality score results suggest
- 14 -
that gDNA purified from the GenTegraTM product stored under all treatment
conditions behaves similarly to cell line gDNA stored in best-case conditions
at -20°C.
Conclusions
Taken together, consistency of HLA genotypes from identically sourced
samples and lack of statistically significant difference in average quality score
for stored samples compared to controls, validate the integrity of gDNA
purified from the GenTegraTM product matrix after all tested treatment
conditions, including the worst-case, 56°C group.
High molecular weight genomic DNA purified from the GenTegra TM product
matrix was used to successfully genotype the most polymorphic genes in the
human genome. Next generation HLA genotyping was chosen to test the
integrity of GenTegraTM product matrix treated genomic DNA due to the
requirment for high-quality, long clonal sequencing reads. Results of this test
demonstrate the efficacy of the product as a suitable genomic DNA
preservation tool for collection, long-term biobanking, or shipping of DNA at
fluctuating and high temperatures. Thus, implementation of the GenTegraTM
product matrix should
greatly reduce sample
compromising data integrity.
- 15 -
storage
cost
without
Abbreviations
NGS: Next generation sequencing
SBT: Sequence based genotyping
gDNA: Genomic deoxyribonucleic acid
HLA: Human leukocyte antigen
PCR: Polymerase chain reaction
qPCR: Quantitative ploymerase chain reaction
Competing Interests
MEH and LYW are employees of GenTegra LLC. GenTegra LLC reimbursed
the laboratory at Children’s Hospital Oakland Research Institute for the cost of
reported HLA genotyping experiments.
Authors' contributions
MEH initiated and conceptualized the project, provided project funding, and
edited the manuscript. LYW carried out DNA preservation laboratory work and
DNA integrity tests, and participated in project design and manuscript
preparation. DJP carried out and reported statistical analysis of sequence
quality and participated in manuscript preparation. SLM carried out HLA
genotyping laboratory work, participated in project design, advised data
analysis, and was the primary manuscript author. JAN helped design and
direct the project and edited the manuscript. All authors read and approved
the final manuscript.
Author Information
MEH is an expert and pioneer in DNA dry storage methodology. He was a cofounder and Chief Scientific Officer for GenVault and now serves as Chief
- 16 -
Sceintific Officer at GenTegra LLC. JAN is an internationally regonized expert
in HLA genetics.
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Figure Legends
Figure 1. A. DNA Storage Timecourse. Either 3 g or 5 g of purified gDNA
sample was treated with GenTegraTM DNA stabilization matrix and stored at
either ~25°C, 37°C, or 56°C for 7 months followed by an incubation at
ambient temperature (~25°C) for a total period of 4 years. B. Native Agarose
Gel Assessment of gDNA Quality. Subsequent to the 4 year storage, the 3
g and 5 g samples were rehydrated and subjected to native 0.8% agarose
gel electrophoresis with ethidium staining. A (collapsed) DNA band with an
apparent molecular weight of 40 kb indicates that the average duplex DNA
strand length for such samples is in excess of approximately 40 kb. C. PCR
Assessment of DNA Quality. 25 ng of each stored DNA sample was
subjected to quantitative real-time PCR (qPCR) employing a 2.5 kb region of
mitochondrial DNA as the target. qPCR was performed in triplicate and the
average CT values for each sample are plotted relative to CTs obtained for
25ng of a highly validated control DNA (Roche Applied Sciences).
Figure 2. HLA-A exon 4 Amplicon Fragment Analyzer Trace Overlay.
Capillary electrophoresis was performed to visualize the HLA-A exon 4
amplicon size range distribution among all samples and controls. Upper and
lower markers used for size determination are visible at positions of 35 base
pairs (bp) and 1500 bp, respectively. Samples represented within the upper
frame were amplified together on a PCR assay plate with the control (PTC)
noted in the frame. Samples represented within the lower frame were
amplified together on a separate PCR assay plate with the control (PTC)
noted in the frame.
- 21 -
Figure 3. Quality Score Distributions. A-C, average quality scores across
all reads at indicated nucleotide position for each sample. A, from sequencing
run 1; B, from sequencing run 2; and C, from sequencing run 3. D. Box plots
of the quality score distributions for all reads at nucleotide position 423.
Replicates were averaged and median centered.
Figure 4: Quality Score Average Comparisons. For each set of conditions,
averages (mean, median, mode) were measured across all reads within a
replicate and all sample replicates (n = 3).
- 22 -
Degrees Celsius
A
25C (C1-C3; D1-D3)
37C (C4-C6; D4-D6)
56C (C7-C9; D7-D9)
60
40
Stored gDNA Amount:
C1-C9 = 3 micrograms
D1-D9 = 5 micrograms
20
0
1
0
2
3
4
ladder D1 D2
40,000
5090/
5000
5090/
5000
C9
C8
C7
C6
C5
C4
C3
C2
C1
40
35
30
25
20
15
Control DNA
Average CT C
25C 25C 25C 37C 37C 37C 56C 56C 56C
Average CT 40,000
D3 D4
D5
D6
D7
D8
D9
40
35
30
25
20
15
D9
C9
D8
C8
D7
C7
D6
C6
D5
C5
D4
C4
D3
C3
D1
C2
Control DNA
ladder C1
B
D2
Years
25C 25C 25C 37C 37C 37C 56C 56C 56C
Lower Marker
HLA-A Exon 4 Amplicon
Upper Marker
A
C
Run 1
Run 3
B
D
Run 2
40 Average Quality Score 35 30 25 Qavg 20 Qmed 15 Qmode 10 5 0 Storage Temp 25°C DNA 3 ug 37°C 56°C 25°C 37°C 56°C 3 ug 3 ug 5 ug 5 ug 5 ug Sample Treatment CondiAons PTC