AmpliSeq™ is a next generation sequencing library preparation method for targeted re-sequencing that utilizes highly multiplexed PCR to amplify regions of interest. A key to successful AmpliSeq libraries is the primer panel used for target amplification. Until now primers have been available as pre-assembled ready-to-use panels, or as custom made-to-order panels. We describe a new process for creating customized panels consisting of optimized and verified PCR primers. The primer sets are available as whole genes (i.e., all of the primers needed to create libraries that cover the entire coding regions of genes) and are selectable on the ampliseq.com website by either uploading gene lists or choosing genes from disease research areas. We show NGS sequencing data from 10 disease research-oriented panels, including newborn screening research and inherited cancer research, assembled from individual pre-verified gene sets. Panel performance data include coverage uniformity, reproducibility, and sensitivity and positive predictive value of variant calling. To demonstrate flexibility of panel content and performance, the coverage uniformity of the 59 genes recommended by the American College of Medical Genetics and Genomics for reporting of incidental findings (ACMG59) was evaluated by themselves and with up to 135 additional genes and shown to be ≥ 97% in all contexts. We also demonstrate the robustness of this method using a variety of sample types (fresh, frozen, and dried blood, cheek swabs) with both manual and fully automated library preparation methods. For Research use only. Not for use in diagnostic procedures.

1. Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com
S Roman1, D Mandelman1, A Broomer1, T Biorac1, J Kilzer1, S Rozenzhak, X Duan1, M Allen1, F Hyland2, M Manivannan2, Y Fu1, Y Zhu1, E Williams1, A Hatch1, Y Tian1, A Kothandaraman2, C Van
Loy1, A Arias1, S Sovan3, J Chang1, M Andersen1
A novel method for building custom AmpliSeq™ panels using optimized PCR
primers
Tables showing average True Positive (TP), False Positive (FP),
and False Negative (FN) calls for single nucleotide polymorphisms
(SNPs) for all libraries prepared for NA12878 (top) and NA24385
(bottom). SNP Sensitivity (Sens.), SNP Positive Predictive Value
(PPV), and Combined (SNPs + insertions/deletions) Sensitivity and
Positive Predictive Value were calculated. Sensitivity = TP/(TP
+FN); PPV = TP/(TP+FP). Note that SNP Sens. and PPV for
ACMG genes alone or combined with NB or Hem genes were very
consistent, demonstrating the robust performance of on-demand
gene designs in varied environments.
Gene Base Uniformity (GBU) is the % of CDS bases (coding DNA
sequence, the portion of a gene's DNA that codes for protein - also
called exons - plus 5 base padding) covered at ≥ 0.2X mean CDS
coverage for a given gene. Plot shows aggregate GBU CVs for all
genes, all panels, and all samples tested, demonstrating high
reproducibility of gene performance.
Figure 1. Panel Base Uniformities – All
Samples and All Methods
ABSTRACT
AmpliSeq™ is a next generation sequencing library
preparation method for targeted re-sequencing that utilizes
highly multiplexed PCR to amplify regions of interest. A key to
successful AmpliSeq libraries is the primer panel used for
target amplification. Until now primers have been available as
pre-assembled ready-to-use panels, or as custom made-to-
order panels. We describe a new process for creating
customized panels consisting of optimized and verified PCR
primers. The primer sets are available as whole genes (i.e., all
of the primers needed to create libraries that cover the entire
coding regions of genes) and are selectable on the
ampliseq.com website by either uploading gene lists or
choosing genes from disease research areas.
We show NGS sequencing data from 10 disease research-
oriented panels, including newborn screening research and
inherited cancer research, assembled from individual pre-
verified gene sets. Panel performance data include coverage
uniformity, reproducibility, and sensitivity and positive
predictive value of variant calling. To demonstrate flexibility of
panel content and performance, the coverage uniformity of
the 59 genes recommended by the American College of
Medical Genetics and Genomics for reporting of incidental
findings (ACMG59) was evaluated by themselves and with up
to 135 additional genes and shown to be ≥ 97% in all
contexts. We also demonstrate the robustness of this method
using a variety of sample types (fresh, frozen, and dried
blood, cheek swabs) with both manual and fully automated
library preparation methods. For Research use only. Not for
use in diagnostic procedures.
INTRODUCTION
Next generation sequencing (NGS) refers to the parallel
sequencing of hundreds to millions of DNA fragments. The
consequent increase in throughput and decrease in price has
led to an explosion of genetic information for a variety of
organisms. Ion AmpliSeq™ library preparation is a
multiplexed PCR-based method for amplifying and preparing
specific regions of interest in a genome for sequencing on Ion
Torrent™ NGS instruments. This library preparation method
can accommodate from dozens to many thousands of primer
pairs in single tube reactions, and can utilize from one to
many primer pools for maximum flexibility. Primers are
available as pre-assembled ready-to-use panels or as custom
made-to-order panels.
Ion AmpliSeq™ On-Demand panels have been developed to
meet the rapidly changing needs of human inherited disease
research. On-demand panels are unique in that researchers
can upload genes of interest or design panels using a content
selection engine (accessible via www.ampliSeq.com) based
on disease research hierarchies. In addition, the genes
available in the on-demand catalog comprise optimized and
wet-lab verified primers. Predicted coverage of catalog genes
based on wet lab data is displayed in the panel design
interface. On-demand panels are compatible with all Ion
Torrent™ library preparation, templating, and sequencing
methods.
In this poster, we show data from on-demand panels drawn
from 10 disease research areas. The data demonstrate that
on-demand panels yield high base uniformity, can be used
with a variety of sample types and library preparation
methods, and that genes run in different panels perform
similarly across the different environments.
MATERIALS AND METHODS
Samples and Sample Preparation. NA12878 and NA24385
were obtained from the Corielle repository (Coriell Institute for
Medical Research, Camden NJ, USA). Genomic DNA (gDNA)
from fresh and frozen blood samples was purified with the
iPrep™ PureLink gDNA Blood Kit (Thermo Fisher Scientific,
Waltham MA, USA, cat no. IS10005) on the iPrep™
Purification Instrument. gDNA from buccal swabs was purified
using the iPrep ChargeSwitch™ Buccal Cell Kit (Thermo
Fisher Scientific, cat no. IS10003) on the iPrep™ Purification
Instrument. Dried blood punches were prepared from the
frozen blood samples as follows: 100 µL of the thawed blood
was applied to FTA paper (GE Healthcare, Chicago IL, USA,
cat no. WB120055). After drying at room termperature for 4
hours, 1.2 mm paper samples were punched from the FTA
paper using a unicorn punch (GE Healthcare, cat no.
WB100028). To use a blood punch directly in the AmpliSeq™
pre-amp reaction, the punches were placed in PCR strip
tubes and treated twice, five minutes per treatment, in 200 µL
FTA purification reagent (GE Healthcare, cat no. WB120204).
Then the punches were rinsed twice in 200 µL low TE (10 mM
Tris-HCl, pH 8.0, 0.5mM EDTA), 5 minutes per rinse. After the
final rinse, all the TE was removed and modified Ion AmpliSeq
pre-amp reaction mixes were added directly to the punches
and used immediately in PCR.
Ion AmpliSeq™ On-Demand Panels. Panels from ten
disease research areas were designed and ordered using the
content selection engine on www.ampliSeq.com (Thermo
Fisher Scientific). For details on the content selection engine,
see ESHG Poster P16.60D: “Developing custom next-
generation sequencing panels using pre-optimized assays: an
integrated approach from disease research area to
functionally annotated variants”.
Library preparation and sequencing. Ion AmpliSeq
libraries were made either manually using the Ion AmpliSeq
Library Kit Plus (Thermo Fisher Scientific, cat. no. 4488990)
or automated on the Ion Chef™ Instrument (cat. no. 4484177)
using the Ion AmpliSeq™ Kit for Chef™ DL8 (cat no. A29024).
Manual libraries were quantified using the Ion Library
TaqMan™ Quantification kit (cat no. 4468802). Barcoded
adapters (cat. no. 4474517) were used to enable pooling of
different samples on sequencing chips.
Libraries were templated and loaded onto Ion S5™ 530™
chips using the Ion 530™ Kit (cat. no. A30010) on the Ion
Chef™ Instrument (cat. no. 4484177). Sequencing was
performed on the Ion S5™ System (cat. no. A27212). Torrent
Suite™ software (v5.4.0) was used to plan sequencing runs
and analyze data. Coverage analysis (v4.0.5) and Variant
Caller (v5.4.0.46) used to evaluate panel uniformity and
sample variants, respectively. Validate Variant Caller
(v0.2.1.1) was used to calculate Sensitivity and Positive
Predictive Value for NA12878 and NA24385 samples.
1 Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA, USA; 2 Thermo Fisher Scientific, 180 Oyster Point Blvd, South San Francisco, CA , USA; 3 Thermo Fisher Scientific, 6055 Sunol
Blvd, Pleasanton, CA, USA
Panel Base Uniformity: % of bases in target with read depth ≥
0.2X of the mean read depth for all panels, all samples, and all
library preparation methods tested with a given panel. Error bars
represent ± 1 SD.
Figure 7. Performance of ACMG Gene
Designs in Different Backgrounds
Figure 3. Panel Base Uniformities –
Automated vs. Manual Library Prep
Panel Base Uniformity: same as Fig. 4; uniformity of libraries
prepared by the Ion Chef™ (blue bars) compared to libraries
prepared by the manual AmpliSeq method (red bars). Error bars
represent ± 1 SD.
Table 1. Disease Research Panels, Samples Tested, and Library Preparation Methods
Figure 2. Panel % Bases On Target – All
Samples and All Methods
Panel % Bases On Target: % of bases in sequencing reads that
map to HG19 genome and also map to target regions for all
panels, all samples, and all library preparation methods tested
with a given panel. Error bars represent ± 1 SD.
Panel Base Uniformity: same as Fig. 4; uniformities of libraries
prepared from matched frozen (darker bars) and dried blood
samples (lighter bars) for two panels. Sample 1 data are shown
with red bars, Sample 2 data are shown with blue bars. See
Materials and Methods for details of sample prep for blood and
dried blood punches. Error bars represent ± 1 SD.
Figure 4. Panel Base Uniformities – 2 Panel,
Matched Frozen and Dried Blood Samples
Figure 5. Reproducibility of Gene Uniformity
across all genes, libraries, and panels
Log2 ratio plots. Top: GBU ratios for 59 ACMG genes run as a
stand alone panel (denominator in ratio calculations), with the 136
gene in the Hematology panel (numerator in blue dot data points),
or with the 76 genes in the Newborn Screening panel (numerator in
red dot data points). The gene furthest from ideal (Log2 = -0.222)
was TNNI3, with GBU = 0.992 in ACMG alone, and GBU = 0.851 in
ACMG+Hem. Ratios for 59/59 and 58/59 ACMG genes were
between 0.9 – 1.1 (log2 = -0.16 – 0.14) in the NB and Hem
backgrounds, respectively.
Bottom: GBU ratios for Hem (green dots) and NB (magenta dots)
genes when run alone (denominator) or in combination with 59
ACMG genes (numerator). The gene furthest from ideal (Log2 =
-0.357) was CYBA, with GBU = 0.984 in Hem alone, and GBU =
0.768 in ACMG+Hem. Ratios for 75/76 NB genes and 134/136
Heme genes were between 0.9 and 1.1 (log2 = -0.16 – 0.14).
Table summarizing panels and samples tested. Disease Research Area panels were designed by selecting research areas and/or specific
diseases with the content selection engine on www.ampliSeq.com. For more details on the Gene-Disease associations utilized by this
content selection engine, see poster P16.60D: “Developing custom next-generation sequencing panels using pre-optimized assays: an
integrated approach from disease research area to functionally annotated variants”
Panels tested are summarized in the table. “Amplicons” refers to the number of individual target molecules that are required to tile across
coding regions of genes and are amplified in the library. Chef and Manual refer to Ion Chef™ automated library preparation and manual
library preparation, respectively. “ref gDNAs” refers to the two reference genomic DNA samples, NA12878 and NA24385, from the Coriell
Institute for Medical Research.
Table 2. Variant Calling by Panel for Reference
Genome Samples
RESULTS
CONCLUSIONS
Ion AmpliSeq™ On-Demand panels are a powerful new tool
for human inherited disease genetic research. Primers for
over 1000 genes with strong links to inherited diseases are
available now and the catalog will soon grow to ~5000 genes.
Genes from the on-demand catalog can be combined in
custom combinations to fit specific research goals.
We have shown performance of a variety of disease research
panels ranging from 17 to 300 genes. Panel base uniformity
was ≥ 95% and typically ≥ 98% (Fig 1). We showed that the
panels are compatible with automated and manual library
preparation methods (Fig. 2), and with a variety of input
samples, including dried blood punches added directly to the
initial target amplification reaction (Fig. 4). Our data also
show that Gene Base Uniformity across all library prep
methods, sample types, and panels was very consistent (Fig.
5).
We have also shown that the performance of a core group of
59 genes (recommended by the American College of Medical
Genetics and Genomics for reporting of incidental findings)
performed very consistently when tested alone and in
combination with genes from other panels (Figs. 6 & 7). This
demonstrates the robustness of this technology for
customizing panel content while maintaining performance.
Finally, for two reference genomes (NA12878 and NA24385),
we have shown high Specificity and Positive Predictive Value
for SNP and SNP+indel variant calling for all panels tested
(Table 2).
Ion AmpliSeq™ On-Demand panels are expected to
accelerate the pace of human genetic research.
For Research use only. Not for use in diagnostic
procedures.
Figure 6. Performance of ACMG gene Designs
in Different Backgrounds
Panel Base Uniformity: same as Fig. 4; uniformity of 59 ACMG
genes in libraries prepared by all methods for all samples tested
with ACMG, ACMG+Hem, and ACMG+NB panels. Data show that
overall panel uniformity for the 59 ACMG genes is consistently
above 97% for these 59 genes alone or in combination with other
content. Blue line depicts 59 gene panel uniformity; red line
depicts 97% uniformity.