Illumina Interview: Robert Daber

Personalized Cancer Therapies through Precision
Diagnostics
Dr. Robert Daber uses TruSeq® Custom Amplicon to conduct genomic testing that can informprognosis and therapeutic choices.
Dr. Robert Daber is Technical Director of Clinical Genomics at Penn
Medicine’s Center for Personalized Diagnostics (CPD). He is a boardcertified clinical cytogeneticist with expertise in bioinformatics, SNP
arrays, and next-generation sequencing (NGS). He oversees theclinical diagnostics laboratory, using NGS to identify distinct molecularchanges and genomic signatures in the DNA of tumor specimens toprovide treating oncologists with the knowledge they need to besttreat their patient’s disease.
Q: What are the goals of the Center for Personalized Diagnostics?
Robert Daber (RD): Our goal at the CPD is to identify as many clinically actionable mutations that are present in a tumor specimen as
possible so that clinicians can better treat their patient’s disease, either
by understanding the prognosis, or by identifying appropriate clinical
trials or approved targeted therapies.
Q: What applications are you using TruSeq Custom Amplicon for?
RD: We used TruSeq Custom Amplicon to develop a liquid tumor panel,
our hematologic malignancy panel. It was designed primarily to identify
important molecular changes across genes previously studied in acute
myeloid leukemia (AML). A seminal paper was published in the New
England Journal of Medicine in early 2012 that demonstrated the importance of 16 genes in this disease. As a result, we designed our custom
panel and offered it as both a clinical test and research tool to clinicians
at our institution. Since February, we’ve analyzed over 150 AML patientsclinically and have found the assay to be very robust. We have also hada few research projects use this panel. The capture reproducibility is so
robust that we have been able to design copy number calling algorithms to identify chromosomal imbalance in the regions interrogatedby the panel, including being able to call gender on each sample. We
have amplicons on the sex chromosomes and we’re able to quantitate
dosage and determine if it’s male or female. This provides us with a nice
QC metric after performing a run, to ensure that there were no sample
or plate mix-ups during processing. That’s really nice in a clinical setting.
We have also had incredible sensitivity with detecting both point mutations and insertions/deletions with this assay. In addition to this panel,
we have a second version with expanded content under validation, as
well as a genome-wide copy number panel we’ve been calling SNPseq.

“The lowest [input amount] we’ve
used is 8 ng total DNA and theresults were beautiful.”

Robert Daber, Ph.D., is Technical Director of Clinical Genomics atPenn Medicine’s Center for Personalized Diagnostics.

Q: What other amplicon panels are you using?
RD: Aside from our Hematologic malignancy panel, which includes
33 genes, we have also validated the TruSeq Amplicon - Cancer Panelfor use on all of our solid tumors. We’re validating our second custom
panel, which is an expanded heme malignancy panel with 68 genesthat addresses acute myeloid leukemia (AML), myelodysplastic
syndrome (MDS), myeloproliferative neoplasms (MPN), and chronic
lymphocytic leukemia (CLL). A single-tube, ~3,700 amplicon assay*is also under development. This assay interrogates the genotype ofevenly spaced single nucleotide polymorphisms (SNPs) across thegenome to provide us with genome-wide copy number coverage.
Q: Why did you choose TruSeq Custom Amplicon?
RD: TruSeq Custom Amplicon allowed us to build our own hematologic malignancy assay, and the off-the-shelf TruSeq Amplicon Cancer Panel addressed our solid tumor needs.
Q: How has TruSeq Custom Amplicon changed what you areable to do in your laboratory?
RD: The biggest impact is that it’s a one-day protocol. From aworkflow perspective, in one working day, a technician can analyze
anywhere from 1 to 96 samples. We try to keep batches under 48,
but we’ve successfully generated 48 libraries in a single day without* This amplicon panel is part of an ongoing research collaboration. TSCA supports custom designs up to 1,536-plex.

Illumina Interview: Robert Daberany problems. This has allowed us to tackle larger projects with a few
hundred samples, and turnaround results fairly quickly. On the clinicalside, we’ve been able to validate these assays and offer them asclinical tests, running nearly 600 clinical samples since February. As
the demand continues to grow, we have plenty of margin for growth
because the assay is so scalable. There’s really no extra steps when
you add more samples to a run plate until you pass the 96 sample
mark in a day. At that point, the bottleneck becomes reviewing thedata and generating reports. With the genes interrogated across ourtwo panels, we are finding disease associated mutations in about 75%of the samples we test. That’s a fairly high yield.

that have a hard time mapping with routine alignment software. We
sequence 186 base pairs in both directions in our assay and struggle
with finding larger indels when processing that data with standard
analysis pipelines. Due to the nature of the TruSeq Custom Amplicon
assay, and having the primer sequence at the beginning of every
read, we can go through and find those mutations using software
we have developed called Garbage Picker. This program effectively
searches through the trash, or the data that cannot be mapped
properly to the human genome due to the large alterations in thesequence reads, and flags those amplicons with enrichment overnormal background noise.

Q: What do you look for in a new assay and how did TruSeq
Custom Amplicon measure up?

Q: What’s the smallest amount of DNA that you’ve tried withgood results?

RD: First, we look for coverage of desired regions. If we can’t capture
what we want to sequence, then the assay isn’t going to work for us.
Using TruSeq Custom Amplicon, we captured over 95% of our desired
target region. This was achieved by sequencing to a minimum mean
depth of coverage of 1,000×, but on average we’ve been doing somewhere between 2,000× and 4,000×.

RD: The lowest we’ve used is 8 ng total DNA and the results werebeautiful. We’ve analyzed 23 samples where we used less than 25
ng of total DNA and another 15 where we used less than 100 ng ofinput DNA and have had beautiful results. The assay can perform very
well on low input amounts, but it’s critical to understand the quality ofthe DNA. Good quality DNA as determined by our QC assay will havesuccess even at low input amounts. Poor quality DNA will fail even if
over 250 ng is used.

Second, the assay needs to be performed in one day in our clinical setting, and ideally scalable to a large number of samples in that
one-day period. With TruSeq Custom Amplicon, we can scale to 96
samples in a day.
Next, we look for the percent of usable data after running the assay,
sequencing, and filtering the off-target reads and low-quality data.
With our optimized protocol, we’re hitting over 90% useable data on aregular basis, with over 95% of reads on target.

Q: Did TruSeq Custom Amplicon meet your expectations?
RD: Yes. As a clinical lab, we have high standards for performance.
If we don’t have high accuracy, reproducibility, sensitivity, andspecificity, we can’t offer it as a test. We really need something thatis reproducible and robust if we are going to offer it with the potentialof helping to inform patient care.

Lastly, it comes down to reproducibility of mutation capture. We use acustomized informatics solution that incorporates both DNA and library
QC with our informatics. In terms of mutation detection, it is incrediblyreproducible. We’ve performed over 40 clinical runs with the same
positive control and no template control and we’ve had 100% mutation concordance for that positive control in every repeat analysis. Thisis with calling variants down to a 5% allele frequency burden.

Q: What are your future plans for the TruSeq Custom Amplicon?

Q: What have you been able to accomplish with TruSeq Custom
Amplicon?

RD: QC, QC, QC. This is a robust assay, but you need to understandthe quality of the DNA you are putting into the assay. Understanding
how well the DNA will amplify, specifically for samples that were fixedin formalin, is critical to achieving robust performance. We perform
two QC assays on every sample before starting an assay, and thishas really empowered us to push the technology beyond its statedlimitations. You can have very reproducible results if you’re notanalyzing poor quality samples.

RD: The nature of the assays, having the same starting and ending
sequence for every amplicon, allows us to write some robust custom
programs and identify large (10–90 base pair) insertions and deletions“As a clinical lab, we have high standards for performance. If we don’t
have high accuracy, reproducibility,sensitivity, and specificity, we can’t
offer it as a test.”

RD: We have a few larger panels with expanded content either invalidation or under active development. We want to push this assay toits limits.
Q: What advice do you have for other researchers considering
TruSeq Custom Amplicon?

Learn more about TruSeq Custom Amplicon atwww.illumina.com/TSCA.

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Pub. No. 776-2013-039 Current as of 02 December 2013