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Comparing DNA Extraction Methods for the LymphoTrack IVD TRG AssayTT054

Maggie Kaminsky, Brandon Givens, Edgar Vigil, Veronika Atkinson, Emily Finnegan, Ying Huang and Jeffrey E. Miller
Invivoscribe, Inc., San Diego, USAResults

Introduction
The LymphoTrack IVD TRG Assay is an investigational sequencing based assay being validated to identify clonal TRG V-J rearrangements, the associated V-J region DNA sequences and toprovide the frequency distribution of the V-J segment utilization. The assay tests DNA extracted from peripheral blood (PB) to determine the presence or absence of T-cell clonality insubjects. DNA is amplified via PCR using individual master mixes with primers that target family-specific V and conserved J regions of the TRG gene and incorporate adapter sequences withup to 24 individual barcodes. Isolation of high-quality, inhibitor-free, double stranded DNA from peripheral blood samples is essential for successful PCR followed by sequencing. In order toprovide users with variety of DNA isolation methods, 3 commercially available DNA extraction kits were evaluated for use with our LymphoTrack IVD TRG Assay.

• DNA yield and amplicon concentration.
DNA yield was quantified using Qubit dSDNA BR Assay kit. Amplicon concentration determined by the Agilent Bioanalyzer using DNA1000 kit. Datasummarized in Table 1 below.
Table 1. Efficiency with respect of DNA Yield. Average Amplicon Concentration per Extraction Methods

Materials and Methods• The LymphoTrack IVD TRG Assay is being evaluated for identification of clonality in TRG gene rearrangements in individuals with suspect T-cell clonality. The assay targets the human Tcell receptor gamma (TRG) gene locus. The PCR primers are designed to target (V) genes (Family I, II, III, and IV), and the joining (J) gene segments where gene rearrangement occurs
during T-cell development (Figure 1).

Extraction Method

Extraction
input (µL)

Cells input
per oneExtraction

DNA Yield after
Extraction(DNA ng)

Extraction
Efficiency (%)

Avg. ampliconconcentration(nm) N=168

Silica column

200

1.00E+06

2346

36.1

23.2

Precipitation

300

1.52E+06

6209

63.0

20.6

Mag Beads

200

1.00E+06

3292

50.6

20.0• Sequencing results (% Reads).
Each extraction method consistently detected expected TRG V-J rearrangements associated with V-J region DNA sequences. The distribution of % Reads pereach clonal low positive contrived samples V-J rearrangements and negative PB samples was similar between the 3 extraction methods as shown in Figure 4.

Figure 1. Schematic diagram of the human T-cell receptor gamma gene
• The sample type for the LymphoTrack IVD TRG Assay is DNA extracted from peripheral whole blood. DNA is amplified via PCR; PCR products are quantified, pooled, and loaded into aMiSeqDx cartridge which contains all of the reagents required for cluster generation and Sequencing by Synthesis. The sequence data is compiled by the MiSeqDx Instrument into FASTQ
files. Invivoscribe’s LymphoTrack IVD Software is intended to perform automated data extraction, analysis, and generate reports from the input FASTQ files for clonality determination
using the LymphoTrack IVD TRG Assay – MiSeqDx. Figure 2 below represents workflow summary.

DNA
Extraction

DNA
Quant.

Ampliconpurification

PCR

AmpliconQuant.

PoolingLobrary

MiSeq Run

DataAnalysis

Figure 2. LymphoTrack IVD TRG Assay – MiSeqDx Workflow summary• Three commercially available DNA extraction kits were evaluated in this study: (1) representing magnetic beads using QIAGEN MagAttract HMW DNA kit, (2) precipitation using JetFlex
Genomic DNA Purification kit and (3) silica column using QIAGEN, QIACube method. Each kit was tested twice to extract DNA from 3 TRG clonal low positive contrived samples (IVS-0053;IVS-0061; IVS-0075) made by blending clonal cells of known cell input with pooled negative peripheral blood and 3 TRG clonal negative peripheral blood samples (PB1; PB2; PB3) by 2
operators. Each DNA sample was tested by the LymphoTrack IVD TRG Assay in duplicate (TRG Run) and sequenced together on a single MiSeqDx instrument. The study design isillustrated in Figure 3.
Each Extraction Method

Operator 1

Operator 2

Extraction repeat 1

Extraction repeat 2

Extraction repeat 1

Extraction repeat 2

TRG

TRG

TRG

TRG

TRG

TRG

TRG

TRG

Run 1

Run 2

Run 1

Run 2

Run 1Run 2

Run 1

Run 2

Figure 3. Flowchart of Extraction Methods Study• Total DNA yield, quantified LymphoTrack IVD TRG Assay PCR product and final clonal results (%Reads) were compared across the 3 extraction methods.

Figure 4. Distribution of %Reads for the 3 DNA extraction methods (low positive contrived samples and healthy PB samples)

Discussion and Conclusions• All 3 evaluated DNA extraction kits provided enough DNA (≥10 ng/µL) for the LymphoTrack IVD TRG Assay. The precipitation method (PPT) produced the highest total DNA yield while thecolumn method produced the least amount of DNA. Data is presented in Table 1.
• The generated PCR product yields were comparable among these 3 extraction methods. Average amplicon concentrations are presented in Table 1. No PCR inhibition was observed forany of the extraction methods.
• The TRG clonality calls generated using the LymphoTrack IVD TRG Assay - MiSeqDx were 100% concordant for all contrived samples processed using all DNA extraction kits (Table 2).
• Variance components analysis was used to estimate the variability of %Reads due to the extraction methods, operator and random error for low positive contrived samples. The
difference among extraction methods contributed no more than 5% of total variability.
• The variability in reported %Reads for positive samples (i.e. frequency of detected TRG rearrangement) was small with overall CV% ranging from 6.5% to 13.6% across all V-Jrearrangements.
• Comparable results were generated using 3 DNA extraction kits with the LymphoTrack IVD TRG Assay.
• This study demonstrated that LymphoTrack IVD TRG Assay performance is independent of DNA extraction method.
MiSeq® is a registered trademark of Illumina, Inc.
* LymphoTrack IVD TRG Assay for Investigational Use Only. The performance characteristics of this product have not been established.

•All samples showed 100% concordance with expected clonality results data summarized in Table 2 below.
Table 2. DNA Extraction Method Results
Extraction Method
Silica column
Precipitation
Magnetic beadSample TypeConcordance

Negative
Low Positive
Negative
Low Positive
Negative
Low Positive

100% (72/72)
100% (96/96)
100% (72/72)
100% (96/96)
100% (72/72)
100% (96/96)

Within-Method
ConcordanceOverall Concordance100% (168/168)
100% (168/168)

100% (504/504)

100% (168/168)

AMP, November 05-09 2019, Baltimore, Maryland , USA