TOMA OS-seq: A high efficiency targeted re-sequencing strategy for detecting gene amplifications, rearrangements,
indels and point mutations in tumor DNA isolated from FFPE solid tumors and plasma.
Yosr Bouhlal, Alexander McKenzie, Austin P. So. TOMA Biosciences Inc., Foster City, CA.
Case 2: Plasma and core biopsy from esophagus (primary)
A.
ABSTRACT
MiSeq 600V3 chemistry: 2x300 PE reads
Input requirements: >10 ng
Clinical sources: FFPE, plasma, FNA
Time to result from sample receipt: 5 days (6 samples)
Critical genetic errors, namely specific mutations, are required for the development and
maintenance of cancer. Molecular diagnostics that leverage the breadth of next-generation
sequencing (NGS) to identify this mutational landscape are therefore becoming an important
clinical tool in the precision treatment of cancer. However, widespread adoption of NGS
technologies in the clinic is hampered by the complexity of the NGS workflow, and the resulting
limits in analytical capabilities arising from noise introduced by poor step-wise yields and bias
from extensive application of PCR.
TOMA
ALT
QUAL
GENE
CHANGE
7
148504716
rs3217095
rs397889421
AG
A
789.73
EZH2
3’-UTR truncation
22
42522613
rs1135840
COSM1599977
G
C
151.03
CYP2D6
S435T
45%
Alteration frequency
40%
Ratio
Case 1: Core needle biopsy from lung (metastatic colon)
35%
30%
25%
20%
15%
1.0
10%
5%
ERBB2
14.0
0%
0.0
Cancer type
12.0
ID
REF
ALT
QUAL
GENE
CHANGE
10
50667105
rs2228529
COSM427630
T
C
125.9
ERCC6
Q1413R
20
54961541
rs2273535
COSM3736282
A
T
263.77
AURKA
F31I
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
-
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
+
10.0
Es
POS
-
CNA data
He
8.0
Deletion
6.0
4.0
B.
H512890
TOMAseq
Digital PCR
1.40
1.59
BRAF
1.05
0.85
CDK4
0.92
1.10
CDK6
1.36
1.35
EGFR
1.31
1.40
FGFR1
0.89
0.97
ERBB2
13.36
14.47
ERBB3
0.94
1.08
JAK2
0.95
0.98
MET
1.29
1.29
SRC
1.63
1.54
VEGFA
0.74
1.02
14.0
POS
ID
REF
ALT
QUAL
GENE
CHANGE
1
98348885
rs1801265
COSM3735989
G
A
375.77
DPYD
C29R
2
29416572
rs1670283
T
C
821.77
ALK
I1461V
10
43620335
rs17158558
COSM3997965
C
T
450.77
RET
R982C
10
50678717
rs2228526
COSM3751837
rs386561694
T
C
121.84
ERCC6
M1097V
12.0
10.0
R2 = 0.9989
slope = 0.9251
8.0
ERCC1
2.0
1.0
0.0
FFPE
6.0
1.2
3.0
0.8
2.0
0.0
0.0
C.
1.6
4.0
0.8
2.0
4.0
6.0
8.0
10.0
1.2
12.0
2.0
1.6
14.0
CHR
POS
ID
REF
ALT
QUAL
GENE
CHANGE
NA
NA
NA
NA
NA
NA
NA
NA
Amplinome
Figure 2. Sequence analysis of DNA (30 ng) purified from fresh frozen core biopsy from lung. A. List of variants of
putative significance called by TOMA OS-seq data analysis pipeline (left panel) with distribution of gene ratios called
across the panel of 96 genes (right panel). ERBB2 (HER2) was identified as amplified at a p < 0.005. B. Comparison of
ratio calls for 12 genes determined with TOMA OS-seq versus a CLIA validated ddPCR test (left panel) showing a high
correlation (R2 = 0.999) between the two orthogonal methods (right panel).
Method: A fresh fine needle biopsy from lung (right, lower lobe nodule) from a de-identified patient was stored in TOMA
storage solution. DNA was then isolated using the Qiagen Blood and Tissue DNA purification kit as described by the
manufacturer. Purified DNA was then sheared to 600 bp and processed using TOMA OS-seq.
// TREATMENT OPTIMIZATION BY MOLECULAR ANALYSIS
3.0
CHR
Ratio
gene
Multiple alterations
B.
0.0
AURKA
Amplification
Ratio
Ratio
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A.
Figure 1. Outline of the TOMA OS-seq protocol. A. OS-seq probes are designed to tile across a region of
interest (ROI) on either strand, and are extended upon hybridization to each strand. Overlapping reads
generated from each strand encompassing the ROI are sequenced to provide a composite of the mutational
landscape for each ROI. B. The TOMA OS-seq protocol leverages highly modularized and streamlined
steps with defined procedural QC points for the conversion of purified DNA to a sequence-able library within
24 hrs.
Data Analysis: Fastq files were aligned to GRCh37.p13 using BWA-MEM 0.7.1. Variants were
called using GATK 2.3.9-lite under GATK standard practices. VCFs were then annotated using
Variation Reporter version 1.4.1.1 (http://www.ncbi.nlm.nih.gov/variation/tools/reporter) to identify
putative variants of significance. A custom python script was used to determine gene amplification
status by aggregating per base coverage metrics (Picard HS metrics) across each gene versus a
reference sample to generate gene ratios.
REF
2.0
TOMA OS-seq
B.
ID
CCND1
2.0
A.
POS
3.0
To address these deficiencies, we introduce TOMA OS-Seq, a complete targeted resequencing
workflow designed specifically for the requirements of the clinical laboratory. Based on oligoselective sequencing (OS-seq), a panel of 96 genes - each with diagnostic or prognostic value can be sequenced end-to-end from as little as 1 ng of input material, enabling the analysis of
DNA purified from fine-needle aspirates and plasma. Utilizing a minimal number of high efficiency
processing steps to create a targeted library, this simple workflow minimizes the requirement for
PCR amplification, preserving the quantitative relationship among the loci interrogated in the
original sample. TOMA OS-Seq is therefore capable of detecting copy number alterations, in
addition to rearrangements/fusions, insertions/deletions, and single nucleotide variants.
CHR
1.0
0.0
cfDNA
Figure 3. Sequence analysis of cell-free DNA purified from plasma (A&C) and FFPE FNA (B). A. Analysis of DNA (14 ng)
purified from plasma following post-radiative treatment with putative variants of significance (upper) and observed distribution of
gene ratios across panel of 96 genes (lower left), identifying CCND1 as amplified at a p < 0.005. Interrogation of the TCGA dataset
(www.cbioportal.com) revealed the highest incidence of CCND1 amplifications in esophageal cancer (lower right). B. Analysis of
DNA (66 ng) purified from fixed relapsed primary tumor tissue and sheared to 600 bp, with list of putative variants of significance
(left panel) and distribution of gene ratios (right panel), identifying ERCC1 as amplified at a p < 0.005. C. Analysis of DNA (230 ng)
from purified from plasma prior to tumor resection, indicating no discernable variants and amplifications within the circulating DNA.
Method: Two aliquots of blood per draw from a research donor diagnosed with esophageal cancer were collected in 10 mL Streck
tubes and processed into plasma using a double centrifugation method. Cell-free DNA was isolated using the Qiagen CNA
purification kit as described by the manufacturer. FFPE primary tissue was purified as described above. Purified DNA (as indicated)
was processed using TOMA OS-seq.