1.
Kate Rhodes, Fiona Hyland, Karel Konvicka, Rajesh Gottimukkala, Gulsah Altun, Chantal Roth, Alain Rico, and Carl Dowds. Life
Technologies, 5791 Van Allen Way, Carlsbad, CA 92008 • www.lifetechnologies.com
RESULTS
Figure 5. Ploidy assessment by low-pass whole-genome
sequencing confirms aCGH results.
A reference set of 34 previously annotated CNV regions was assessed
using the Ion AmpliSeq™ Comprehensive Cancer Panel across 31 cell line
genomes that contain significant chromosomal aberrations. The CDC
Genetic Testing Reference Materials Coordination Program (GeT-RM)
previously characterized this sample set as reference material. The number
and size range of the duplications and deletions as determined by
microarray are listed. Note that the detection and size range of CNVs are
limited by amplicons spanning the known CNVs. We recommended that at
least 10 amplicons cover a region for robust detection of CNVs. The
performance of the CNV detection algorithm was evaluated using the
medium sensitivity setting in Ion Reporter™ Software.
A) The left side of the panel illustrates the visualization of CNVs over the
entire genome in the karyotype view. The deletion CNV (red line) was
detected in a region of chromosome 11 (selected for focus in the right-
hand panel), at the same position as the deletion detected by SNP array.
B) The CNV red line in the “sample ploidy (seg)” track was detected in a
single gene (EXT2), with no evidence of copy number change in
amplicons covering flanking genes. CNVs are visualized in Ion
Reporter™ Software using a custom version of Integrative Genomics
Viewer (IGV), which offers the standard Genome Browser view as well
as a CNV track with normalized coverage track for the test and control
samples. Note that additional tracks also indicate SNVs, indels, and
genes/exons.
Figure 3. Example of a sample (GM22624) with a deletion
CNV on chromosome 11.
Table 2. Karyotypes of the 10 samples tested with a total of 28
previously characterized copy number changes.
(WC = whole chromosome)
ABSTRACT
Ion Torrent™ semiconductor sequencing, combined with Ion
AmpliSeq™ technology, provides simultaneous identification
of copy number variants (CNVs), single nucleotide variants
(SNVs), and small insertions and deletions (indels) from a
research sample by means of a single integrated workflow.
100% of assayed CNV regions (n=34) were detected using a
reference set of 31 samples with known chromosomal
aberrations. Low-pass whole-genome sequencing data, with
approximately 0.01x read coverage, allowed the rapid ≤10
hour analysis of aneuploidies from research samples with
extremely low initial input DNA amounts—even from a single
cell. Using a control set of 10 samples with known
chromosomal aberrations, 100% of the copy number changes
were found, ranging from gains or losses of whole
chromosomes to subchromosomal alterations tens of
megabases (Mb) in size. The Ion PGM™ System minimizes
the high cost and complexity of next-generation sequencing
and, with Ion Reporter™ Software, facilitates user-defined
CNV and aneuploidy detection, with three sensitivity options
so that copy number analysis workflows can be tuned to
achieve desired levels of sensitivity and specificity.
INTRODUCTION
Copy number variants (CNVs) represent a class of genomic
variation in which large regions (> 1 kb) of the genome can be
duplicated (gains) or deleted (losses) (Figure 1A). Inherited
and de novo CNVs have been associated with many disease
conditions, including cancer and genetic disorders. There can
be a variety of phenotypic impacts from CNVs: deletions can
unmask recessive mutations; duplications and deletions can
alter the copy number of dosage-sensitive genes; deletions
can result in position effects that alter the regulation of genes;
and selection of duplicated regions containing gain-of-function
driver mutations can occur.
Genomic copy number imbalances can involve large regions,
ranging from subchromosomal regions to whole
chromosomes, or aneuploidies. Most normal human somatic
cells contain a diploid [2N] set of autosomes (nonsex
chromosomes) and a pair of sex chromosomes. Cells that do
not contain an exact diploid set are termed aneuploid (Figure
1B). Common types of aneuploidy that survive to term are
monosomy (the loss of one chromosome) of the X
chromosome in females—Turner syndrome (45, X)—and
some trisomies, three copies of a given chromosome in a
diploid cell. Live births are possible with trisomies of
autosomes 13 (Patau syndrome), 18 (Edwards syndrome),
and 21 (Down syndrome), as is the presence of extra sex
chromosomes such as in Klinefelter syndrome (47, XXY) and
Triplo-X syndrome (47, XXX).
MATERIALS AND METHODS
In a single-blind retrospective study, the performance of the
algorithm was assessed on 31 archived samples with known
CNVs that were amplified using the Ion AmpliSeq™
Comprehensive Cancer Panel (CCP) primer set and
sequenced on the Ion PGM™ instrument (Figure 2A).
Across the sample set, 34 previously annotated CNV regions
were assessed based on coverage by amplicons in the
~16,000-amplicon CCP panel. Using Ion Reporter™ Software
and applying the medium sensitivity setting, the CNV
algorithm detected 34 of the 34 CNV segments (Table 1).
Figure 3 illustrates an example of a previously annotated
deletion that was detected in a region of chromosome 11 from
sequence read data. This deletion on the short arm of
chromosome 11 deletes the EXT2 gene, seen in Potocki-
Shaffer syndrome (Figure 3B).
Ion Torrent™ semiconductor sequencing and Ion Reporter™
Software deliver the power to detect aneuploidies and
subchromosomal events across all 23 pairs of chromosomes
via a simple, rapid, and integrated workflow (Figure 2B).
Through the use of whole genome amplification (WGA), gDNA
from a small number of cells—even a single cell—can be
used in the Ion PGM™ sequencing workflow. In a single-blind
study, the performance of the aneuploidy workflow was
assessed on 20 samples: 10 normal samples and a reference
set of 10 samples with characterized aneuploidies (Table 2).
Using the default medium sensitivity setting, the CNV
algorithm in Ion Reporter™ Software detected 100% (n=28) of
the copy number changes including a subchromosomal loss
of only 18 Mb on chromosome 3; with no false positives in the
10 reference samples from low-pass whole-genome
sequencing data with approximately 0.01x read coverage.
Figure 4 illustrates a complex karyotype with a monosomy
(loss) of chromosome 13 and trisomies (gains) of
chromosomes 14 and 15. Importantly, the ploidy predictions
by low-pass whole-genome sequencing were 100%
concordant with aCGH results (Figure 5).
CONCLUSIONS
Ion AmpliSeq™ target selection technology and Ion Reporter™
Software, in combination with the Ion PGM™ System, provide a
robust method for the detection of CNVs, SNVs, and indels in a
sample in a single, rapid workflow solution for your research. Using
a tunable software algorithm that can be user-defined, internal
performance testing demonstrated detection of 34 of 34 CNVs when
the Ion AmpliSeq™ CCP primer set was tested on a reference panel
of 31 cell line samples that harbor significant chromosomal
abnormalities. Ion Reporter™ Software, in combination with the Ion
PGM™ System, provides a robust method for the detection of
aneuploidies from research samples with low amounts of input DNA
in a single, rapid workflow solution. Internal performance testing
detected 100% of the 28 known copy number changes present in 10
samples harboring significant chromosomal aberrations and no false
positives when tested on 10 normal samples. Critically, the results
were 100% correlated with results obtained using aCGH. However,
in contrast to aCGH, sample multiplexing allows barcoding of
samples for simultaneous analysis in a single low-pass whole-
genome sequencing run. In this case, 10 samples were rapidly
sequenced on a single Ion 316™ Chip, greatly reducing the cost per
sample to obtain accurate aneuploidy results. In conclusion, the
workflows presented here provide a rapid, accurate, and sensitive
research solution for CNV and aneuploidy detection from
sequencing read data.
ACKNOWLEDGEMENTS
Samples were kindly provided by Dr. Francesco Fiorentino,
GENOMA, Rome, Italy. Samples which aided in the development of
the algorithm were kindly provided by Dr. Dagan Wells, Institute of
Reproductive Sciences, University of Oxford, UK.
REFERENCES
Find out more at:
lifetechnologies.com/aneuploidy
lifetechnologies.com/ampliseq
TRADEMARKS
For Research Use Only. Not for use in diagnostic procedures.
© 2013 Life Technologies Corporation. All rights reserved.
The trademarks mentioned herein are the property of Life
Technologies Corporation and/or its affiliate(s) or their respective
owners.
CNV and aneuploidy detection by
Ion semiconductor sequencing
Life Technologies • 5791 Van Allen Way • Carlsbad, CA 92008 • www.lifetechnologies.com
Sample Karyotype Size detected
1 -1p36.33.p31.1 82 Mb
2 +5q32.qter 24 Mb
3 -13, +14, +15 WC, WC, WC
4 +14, -19, +22 WC, WC, WC
5 -2pter.q34, +11pter.q12.1, -18 207 Mb, 61 Mb, WC
6 -5, -22 WC, WC
7 +1, -3pter.p25.1, -15, -18, -20 WC, 18 Mb, WC, WC, WC
8 +4p16.3.p14, -8q24.12.qter 44 Mb, 24 Mb
9 +10, -14 WC, WC
10 +1, +6, +8, +9, +11, +19 WC, WC, WC, WC, WC, WC
Table 1. Confirmation of CNVs by Ion Torrent™ semiconductor
sequencing.
Microarray Confirmed by sequence reads
Duplications Deletions Duplications Deletions
Number 11 23 11 23
Size
range
(bp)
2.92 x 106 –
1.55 x 108
8.33. x 105 –
1.55 x 108
A) Read coverage (black) across the 22 autosomes and the sex
chromosomes. Changes from normal are illustrated by the red line
showing trisomies (ploidy of 3) of chromosomes 14 and 15 and a
monosomy (ploidy of 1) of chromosome 13.B) aCGH data across the 22
autosomes and the sex chromosomes with changes from baseline
indicating trisomies (ploidy of 3) of chromosomes 14 and 15 and a
monosomy (ploidy of 1) of chromosome 13.
Figure 2. CNV and aneuploidy detection sequencing
workflows
Life Technologies supplies an easy-to-implement, cost-effective, rapid
and scalable CNV and aneuploidy detection workflows for the Ion
PGM™ System with primary data analysis is performed using Torrent
Suite™ Software. A) CNV detection using Ion AmpliSeq ™ DNA panels
that takes <11-hours (3.5-hour library construction, 4-hour template
preparation, 2-4-hour sequencing run, and 1-hour data analysis) from
library to variant-called results with SNV, indel, and CNV polymorphisms
are determined using Ion Reporter™ Software. B) A rapid (<10-hour
workflow) low-pass whole-genome sequencing for aneuploidy detection
(2 hour library construction, 4 hour template preparation, 3 hour
sequence run, and 1 hour data analysis) from library to results.
Following whole-genome amplification (WG A), library construction and
barcoding is performed using the Ion Xpress™ Plus gDNA Fragment
Library Kit and Ion Xpress™ Barcode 1-16 Adapters Kit, respectively.
Aneuploidy status is determined using Ion Reporter™ Software.
B.
B.
A.
A) These variations include deletions, insertions, inversions, copy
number variants, and segmental duplications. B) Most non-sex cells
contain an exact diploid set of chromosomes; genomes that lack or
contain additional chromosomes are termed aneuploid.
Chromosomal gain, trisomies of chromosomes 14 and 15, are
highlighted in blue while a loss of chromosome 13 is indicated in
red.
A.
Figure 1. Structural variations within the human genome
Using Ion Reporter™ Software, the left-hand panel illustrates the
visualization of aneuploidies over the entire genome in the karyotype
view. The loss (monosomy) of chromosome 13 is indicated in red with
gains (trisomies) of chromosomes 14 and 15 indicated in blue. The right-
hand view can be used to zoom in on a chromosome to determine if the
aneuploidy is the loss/gain of a whole chromosome or a deletion or
duplication of a subchromosomal region.
Figure 4. Example of a sample (#3 from Table 2) with trisomies
of chromosomes 14 and 15, and a monosomy of chromosome
13 detected by low-pass whole-genome sequencing..