We have developed a research method to prepare Ion AmpliSeq libraries directly from small amounts of biological samples such as whole blood, saliva, buccal swabs and even FFPE sections. • The direct library prep methods were automated on the Ion Chef reducing user interaction to loading reagents and samples on to the deck of the Ion Chef. • Sequencing metrics were comparable or improved for the samples used directly without purification as compared to purified DNA controls. Additionally, as shown for buccal swab samples, there was no detrimental effect on variant calling results. • Extremely small samples were successfully processed with this method, which reduces the total time from sample to sequence to 24 hours.

1. Cristina Van Loy, Marcela Carvallo, Denise Topacio, Kate Rhodes, Mark Andersen
Thermo Fisher Scientific, Carlsbad, CA, USA
Preparing libraries directly from archived FFPE sections blood, saliva, and
buccal swabs on the Ion Chef
ABSTRACT
• We have developed a research method to
prepare Ion AmpliSeq libraries directly from small
amounts of biological samples such as whole
blood, saliva, buccal swabs and even FFPE
sections.
• The direct library prep methods were automated
on the Ion Chef reducing user interaction to
loading reagents and samples on to the deck of
the Ion Chef.
• Sequencing metrics were comparable or
improved for the samples used directly without
purification as compared to purified DNA controls.
Additionally, as shown for buccal swab samples,
there was no detrimental effect on variant calling
results.
• Extremely small samples were successfully
processed with this method, which reduces the
total time from sample to sequence to 24 hours.
INTRODUCTION
Standard library preparation methods for next-
generation sequencing require purified DNA.
Purification methods are time consuming and may
decrease the yield of nucleic acids. We have developed
a robust protocol for converting saliva, buccal swab,
and blood and formalin-fixed paraffin embedded
samples (FFPE) directly into Ion AmpliSeq™
sequencing libraries without extraction or purification of
nucleic acids. The direct library prep method is resistant
to inhibitors present in biological samples allowing their
direct use in the amplification reaction.
We have implemented an automated library prep
workflow on the Ion Chef system reducing hands-on
time to about 15 minutes. The AmpliSeq-on-Chef
system includes a set of new consumables and
software to prepare up to 8 AmpliSeq libraries in less
than 8 hours.
MATERIALS AND METHODS
Sample Preparation - We evaluated retrospective (or archived)
samples of formalin-fixed paraffin embedded (FFPE) samples,
saliva, buccal swab, and blood using Ion AmpliSeq DNA
research panels with 119, 269 or 1263 assays designed to
known pharmacogenomics or oncogene targets. For FFPE
samples, FFPE tissue sections on glass slides were scraped
using a pipet tip with 30ul of Transfer Solution and put into wells
of a 96-well plate. Buccal swab samples were resuspended in
100ul of Low TE and 2ul of this suspension was used. Saliva
and blood samples were used directly, 1ul of each was used.
Direct Reagent was mixed with each type of sample and
incubated for 5 minutes at room temperature.
Library Preparation
5X Ion AmpliSeq HiFi Mix and 2X or 5X Ion AmpliSeq Primer
Pools were added to the samples mixed with Direct Reagent.
Libraries were generated using the Ion AmpliSeq Library
Preparation Protocol with minor modifications. Targets were
amplified according to the number of cycles recommended for
FFPE DNA depending on the number of primer pairs in the
primer pool used. Library yields were quantified by qPCR.
Templating and Sequencing
All libraries were templated using the reagents and protocols for
the Ion PGM™ Hi-Q™ Template Kit and subsequently
sequenced with the Ion PGM Hi-Q Sequencing Kit on the Ion
Torrent Personal Genome Machine™ (PGM).
Direct from Sample Library prep and Sequencing Workflow
Data Analysis
Ion Reporter
• Detection of variants
• CNV
• Gene expression
• Gene fusions
Sequencing
IonTorrent S5
or
Ion Torrent PGM
RESULTS
Libraries directly from FFPE, swab, saliva or blood
Libraries generated directly from FFPE tissue slices, whole blood, saliva,
and buccal swab samples all resulted in similar or improved sequencing
performance as compared to purified DNA. For the panels tested,
uniformity of base coverage was >94%, base reads on target were
>91%, and target bases with no strand bias was >94%. We were able to
generate and successfully sequence DNA libraries from single FFPE
slices as small as 2 mm X 2 mm X 5 microns and from a single microliter
of archived blood or saliva samples.
CONCLUSIONS
We developed a research method to directly use
biological samples and low input samples for
generating libraries to reduce the time from
sample to analysis on an NGS platform.
Extremely small samples were successfully
processed with this method, which reduces the
total time from sample to sequence to 24 hours.
Sequencing metrics were comparable or
improved for the samples used directly without
purification as compared to purified DNA
controls. Additionally, as shown for buccal swab
samples, there was no detrimental effect on
variant calling results.
For Research Use Only. Not for Diagnostic Use.
© 2016 Thermo Fisher Scientific Inc. All rights reserved. All trademarks are
the property of Thermo Fisher Scientific and its subsidiaries unless
otherwise specified.
Sample
Saliva
Swab
eluate
Whole
blood
FFPE
1 uL
2 uL
1 uL
10-30
uL
Transfer
Solution
Direct
Reagent
AmpliSeq-on-Chef
+
Templating on Chef
Library Prep
FIGURE 1. Ion AmpliSeq direct from biological sample protocol. FFPE samples were scraped from slides, suspended in proprietary Transfer solution
and 10-30 ul aliquots were mixed with Direct Reagent and directly loaded on to the deck of the Ion Chef system with AmpliSeq reagents. For whole blood
and saliva. 1 ul of the sample was mixed with the Direct Reagent and for eluates from swabs, 2 ul was mixed with the Direct Reagent. The Direct
Reagent mixtures were loaded on to the Ion Chef for automated AmpliSeq library preparation. The automated protocol performs targeted amplification,
digestion, ligation, and normalization on 8 samples without any user intervention. Prepared libraries were then clonally amplified and loaded on to the Ion
chip, again, with the Ion Chef. The templated chips were sequenced on an Ion PGM or S5 sequencing system.
0
0.2
0.4
0.6
0.8
1
1.2
Uniformity of
base
coverage
Percent end-
to-end reads
Percent
base reads
on target
Target
bases with
no strand
bias
FIGURE 4. Whole Blood Direct. AmpliSeq libraries
were generated from whole blood samples using a
269 assay DNA panel. These result in similar
sequencing metrics as compared to a high molecular
weight genomic DNA control (10 ng NA12878).
FIGURE 2. FFPE DNA Direct. A. Ampliseq libraries were generated from various FFPE
samples using a 269 assay tumor DNA panel. Sequencing metrics are equivalent or
improved with the direct method. B. Libraries were generated from 4, 16, and 64 mm2 liver,
lung, or colon FFPE tissue. Uniformity of base coverage is equivalent with all tissue sizes.
0.8
0.85
0.9
0.95
1
1.05
Uniformity of base
coverage
Amplicons reading
end-to-end
Percent base reads
on target
Target bases with no
strand bias
gDNAcontrol
FFPEDNA
FFPEDirect
gDNAcontrol
FFPEDNA
FFPEDirect
gDNAcontrol
FFPEDNA
FFPEDirect
gDNAcontrol
FFPEDNA
FFPEDirect
0.5
0.6
0.7
0.8
0.9
1
1.1
Purified DNA 64 mm2 16 mm2 4 mm2
gDNAcontrol
LiverFFPE
Lung1FFPE
Lung2FFPE
ColonFFPE
LiverFFPE
Lung1FFPE
Lung2FFPE
ColonFFPE
LiverFFPE
Lung1FFPE
Lung2FFPE
ColonFFPE
LiverFFPE
Lung1FFPE
Lung2FFPE
ColonFFPE
DNA
Blood
DNA
Blood
DNA
Blood
DNA
Blood
50%
60%
70%
80%
90%
100%
Uniformity of base
coverage
Amplicons reading
end-to-end
Percent base reads
on target
Target bases with
no strand bias
gDNACtrl
BuccalDNA
SalivaDirect
BuccalDirect
gDNACtrl
BuccalDNA
SalivaDirect
BuccalDirect
gDNACtrl
BuccalDNA
SalivaDirect
BuccalDirect
gDNACtrl
BuccalDNA
SalivaDirect
BuccalDirect
FIGURE 3. Buccal Swab and Saliva DNA Direct. Libraries were generated from buccal
swab or saliva using a 269 assay DNA panel. Both types of samples yielded high quality
libraries as compared to purified DNA controls.
FIGURE 5. Ten buccal Swab Direct libraries using
119 and 1263 assay DNA panels showed similar
assay metrics to purified DNA. Additionally, the same
variant calling results were obtained between purified
and direct libraries.
DNA
Direct
DNA
Direct