understanding of the human immune system, and thereby cancer immunology. αβT-cells are the primary constituents of human cell-mediated adaptive immunity. The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript encompassing the variable portion of the rearranged TCRα and TCRβ subunits, which can be read via NGS in a process termed repertoire sequencing. Until now, the main challenge the field faces is the lack of a technology that can provide a contiguous read of 600 bp to minimize the complexity of designing bias-prone primers and informatics challenges of stitching short reads. Here we leverage the long read capability of Ion 530™ chip to comprehensively sequence all three CDR domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire profiling studies. Initial testing with leukocyte total RNA demonstrates that this multiplex PCR assay produced repertoires that were much more similar to data derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This result suggested that the use of long reads minimizes bias by allowing targeting of less variable regions. To further assess the performance of the assay, we designed a model system of 30 plasmid controls containing common human T-cell CDR3 sequences. Each plasmid was amplified individually and sequenced to confirm the detection of a single clonal population. Analytical sensitivity of the assay and accuracy of the accompanied analysis solution were further evaluated by spiking in plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results showed the assay offers linearity over 5 orders of magnitude of decreasing input concentration. In summary, we have demonstrated a NGS workflow for TCRβ sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less than 48 hours.

1. Read Length (bp)
For Research Use Only. Not for use in diagnostic procedures. Thermo Fisher Scientific • 5781 Van Allen Way • Carlsbad, CA 92008 • thermofisher.com
Denise Topacio-Hall1, Tim Looney2, Geoffrey Lowman1, Elizabeth Linch1, Lauren Miller1, Yongming Sun2, Lifeng Lin2, Xinzhan Peng1,
Mark Andersen1, Fiona Hyland2, Ann Mongan2
(1) Thermo Fisher Scientific, 5781 Van Allen Way, Carlsbad, CA 92008 (2) Thermo Fisher Scientific, 180 Oyster Point Blvd. South San Francisco, CA 94080
Sequencing the Human TCRβ Repertoire on the Ion S5TM
Abstract
3567
ABSTRACT
Next-generation Sequencing (NGS) is proving an important tool in increasing
understanding of the human immune system, and thereby cancer immunology.
αβT-cells are the primary constituents of human cell-mediated adaptive immunity.
The antigen specificity of each αβT-cell is encoded in the 500-600 bp transcript
encompassing the variable portion of the rearranged TCRα and TCRβ subunits,
which can be read via NGS in a process termed repertoire sequencing. Until now,
the main challenge the field faces is the lack of a technology that can provide a
contiguous read of 600 bp to minimize the complexity of designing bias-prone
primers and informatics challenges of stitching short reads. Here we leverage the
long read capability of Ion 530™ chip to comprehensively sequence all three CDR
domains of the TCRβ chain. The Ion 530™ chip offers greater than 15 M productive
reads, allowing a multiplex of 2-4 samples with sufficient coverage for most repertoire
profiling studies. Initial testing with leukocyte total RNA demonstrates that this
multiplex PCR assay produced repertoires that were much more similar to data
derived from 5’-RACE protocol than the commonly used BIOMED-2 primer set. This
result suggested that the use of long reads minimizes bias by allowing targeting of
less variable regions. To further assess the performance of the assay, we designed a
model system of 30 plasmid controls containing common human T-cell CDR3
sequences. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. Analytical sensitivity of the assay and
accuracy of the accompanied analysis solution were further evaluated by spiking in
plasmid concentrations from 10 pg to 0.0001 pg (5 million to 50 copies) in a
background of 100 ng cDNA reverse transcribed from leukocyte total RNA. Results
showed the assay offers linearity over 5 orders of magnitude of decreasing input
concentration. In summary, we have demonstrated a NGS workflow for TCRβ
sequencing that offers multiplex flexibility on Ion S5 with sample to answer in less
than 48 hours.
INTRODUCTION
The immune repertoire refers to the collection of immunoglobulins and T-cell
receptors (TCR) that comprise a key part of an individual’s adaptive immune system.
The large diversity found in the repertoire is formed by rearrangement of gDNA into
variable (V), diversity (D), and joining (J) gene-segments, as well as the deletion and
random insertion of nucleotides during the rearrangement process prior to
transcription. The diversity of TCRαβ molecules is estimated to be >1012.1 Molecular
analysis of T-cell receptor genes is widely used to support the final diagnosis of
lymphoproliferations2, and is gaining application in biomarker identification in
immune-oncology. TCR gene rearrangement studies are also useful to evaluate the
clonal relationship between two lymphoid malignancies in one patient and for staging
of the disease2. The Oncomine™ TCR-Seq β assay was developed to target the
variable gene region and the constant gene region, allowing amplification of the full
length TCRβ. The assay includes all variable genes and alleles in the gold-standard
IMGT database, and target framework 1 (FR1) of the V-region producing amplicons
which cover CDR1, 2, and 3.
The Oncomine™ TCR-Seq β assay produces libraries 325-400bp in length.
Together with the S5 530 chip on the Ion S5 Sequencing Platform, >50,000 clones/
sample are generated from up to 16 samples with a workflow under 48 hours. The
output is unbiased, as v-gene primers were optimized to reproduce results from
600bp 5’-RACE protocols. Lineages are reported from highly accurate results with
ultra-low base call error rates after analytics identify and eliminate PCR and
sequencing errors.
We have benchmarked the Oncomine™ TCR-Seq β assay against two most
commonly used approaches: Rapid Amplification of cDNA Ends (5’-RACE) and
multiplex PCR (MPCR) using BIOMED-2 primers. 5’-RACE utilizes one primer set
that targets the known C-gene region of the mRNA transcripts, while the second
general primer is synthetically added to the 3’ end of the cDNA using terminal
deoxynucleotidyl transferase (TdT) and cCTP.3 MPCR with the BIOMED-2 primers
utilizes a pool of primers placed in FR2 and amplifies the CDR3 regions.1,3
To further assess the performance of the Oncomine™ TCR-Seq β assay, a model
system of 30 plasmids was designed against 30 well characterized CDR3 T-cell
lymphomas2. Each plasmid was amplified individually and sequenced to confirm the
detection of a single clonal population. The plasmids were also amplified as pools at
varied concentrations to assess linearity of expected frequencies.
MATERIALS AND METHOD
The Oncomine™ TCR-Seq β assay was developed to target the variable gene region
and the constant gene region, allowing amplification of the full length TCRβ. The
primer set covers all known variable genes and alleles in the gold-standard IMGT
database. The resulting amplicons cover all CDR regions.
The Ion AmpliSeq™ Library Preparation Protocol was used to generate Oncomine™
TCR-Seq β libraries with leukocyte total RNA and 30 plasmids. The 30 plasmids
were manufactured by GeneArt (Thermo Fisher Scientific). They were either left
intact or digested using FastDigest SfiI (Thermo Fisher Scientific). Concentration
was determined by Qubit and the plasmids were serially diluted in Low TE with 0.01
mg/mL glycogen.
High throughput:
>50,000 clones/
sample from blood,
cell populations, or
Fresh-Frozen tissue
generated from a
single tube workflow.
Highly accurate:
sequencing and
amplification errors are
corrected with
statistical models
leveraging TCR mRNA
as input.
Diversity view
V gene
CDR3length(nt)
Size of clones
Clonotype identification
V-gene identification
Readcount
Proportion of different alleles
Analysis solution:
Complete Ion Reporter
analysis solution
including clonotype
identification and
diversity, CDR3 length,
and multisample
analysis for longitudinal
tracking for clones of
interest.
a.) Comparisons of V-gene usage to 5’-RACE
library prepara9on strategies give correla9on
values ranging from r = 0.90-0.92.
b.) Correla9on in V-gene usage between
BIOMED-2 and 5’-RACE are normally in the
range r ~ 0.75-0.80
For Research Use Only. Not for use in diagnostic procedures.
© 2017 Thermo Fisher Scientific Inc. All rights reserved.
All trademarks are the property of Thermo Fisher Scientific
and its subsidiaries unless otherwise specified.
2-day workflow using the OncomineTM TCR-Seq β assay - Immune Repertoire sequencing using AmpliSeqTM library construction
Variable Diversity Joining Constant
N1 N2
A. Adult IGH or TCRBeta chain rearrangement
In adult B and T cells, the process of VDJ rearrangement very often involves
exonucleotide chewback of VDJ genes and the addition of
non-templated bases, forming N1 and N2 regions in the B cell receptor
heavy chain CDR3 and the T cell receptor Beta chain CDR3.
These processes vastly increase IGH and TCRB CDR3 diversity.
Variable Diversity Joining Constant
B. Fetal IGH or TCRBeta chain rearrangement
In the fetus, the process of VDJ rearrangement often occurs without
exonucleotide chewback of VDJ genes and addition of non-templated
bases, resulting in a restricted IGH and TCRB CDR3 repertoire that is
distinct from the adult repertoire.
These structural differences can be used to distinguish fetal B and T cell
CDR3 receptors from maternal B and T cell CDR3 receptors in cell free
DNA present in maternal peripheral blood. In this way, fetal B and T
cell health and development may be monitored in a non-invasive
manner.
Figure 1. Structural differences between fetal and adult B and T cell receptors
Variable Diversity Joining Constant
N1 N2
A. Adult IGH or TCRBeta chain rearrangement
In adult B and T cells, the process of VDJ rearrangement very often involves
exonucleotide chewback of VDJ genes and the addition of
non-templated bases, forming N1 and N2 regions in the B cell receptor
heavy chain CDR3 and the T cell receptor Beta chain CDR3.
These processes vastly increase IGH and TCRB CDR3 diversity.
Variable Diversity Joining Constant
B. Fetal IGH or TCRBeta chain rearrangement
In the fetus, the process of VDJ rearrangement often occurs without
exonucleotide chewback of VDJ genes and addition of non-templated
bases, resulting in a restricted IGH and TCRB CDR3 repertoire that is
distinct from the adult repertoire.
These structural differences can be used to distinguish fetal B and T cell
CDR3 receptors from maternal B and T cell CDR3 receptors in cell free
DNA present in maternal peripheral blood. In this way, fetal B and T
cell health and development may be monitored in a non-invasive
manner.
Figure 1. Structural differences between fetal and adult B and T cell receptors
FR1 FR2
Diversity (D) Joining (J) Constant (C)
Variable gene (V) CDR3
FR3
CDR1
CDR2
Figure 1. Schematic representation of a T-cell receptor which has undergone V(D)J
rearrangement, identifying the CDR1, 2, and 3 regions, and the Framework (FR1, 2, and
3) regions. Using RNA/cDNA as input allows design of primers targeting the (V) and (C)
regions.
5’-RACE
BIOMED-2
5’ RACE
5’ RACE
AmpliSeq
TCRβ
AmpliSeq
TCRβ
0.00001
0.0001
0.001
0.01
0.1
1
0.00001 0.0001 0.001 0.01 0.1 1
Log10ObservedPlasmidFrequency
Plasmid Input (pg)
Frequency vs Plasmid Input
(3) 530 Chips with 10 Individual Plasmids each
50
copies
500
copies
5,000
copies
50,000
copies
5
copies
Standard kit conditions for template preparation and sequencing used the Ion 510™
Kit - Chef and the Ion S5™ Sequencing System, respectively, for evaluation of the
generated libraries. All libraries were sequenced on Ion 530™ Chips. All results were
analyzed using the Ion Torrent™ Browser and the TCRβ Repertoire Plug-in. The Ion
Reporter™ Software is still in development.
RESULTS
Figure 5. Libraries prepared using pools of plasmids with combinations of input
concentrations (10pg to 0.0001pg = 5M to 50 copies) in a background of 100ng
Leukocyte cDNA. a.) Experimental design. Varied concentrations of 30 plasmids in 4
pools. b.) Data showing linearity over 5 orders of magnitude. c.) Pools of 30 plasmids
at equimolar concentrations have plasmid frequencies within one order of magnitude.
Plasmids were either individually digested, digested in bulk, or left intact..
CONCLUSIONS
The Oncomine™ TCR-Seq β assay combined with the Ion S5 sequencing platform is a
rapid method to identify clones from RNA. Using RNA/cDNA input allows coverage of
many productive variations of T-cell receptors. Since sequencing is targeted, there are
low to moderate read output requirements (typically 1-2M reads per sample), resulting in
the identification of >50,000 clones. Since total read counts on an Ion 530™ chip are
typically 15-20M, one can multiplex up to 16 samples.
We have run the same leukocyte total RNA sample in the Oncomine™ TCR-Seq β
assay, 5’-RACE protocol, and BIOMED-2 multiplex PCR protocol. The Oncomine™
TCR-Seq β assay correlates to 5’-RACE well (r=0.90-0.92). This is better than the
BIOMED-2 correlation to 5’-RACE (r=0.75-0.80). Comparable library preparation results
do not guarantee comparable overall results, since our error model will likely disqualify
more reads generated by this method. So, additional testing was performed with a
model system of 30 plasmids. These plasmids were designed against 30 well
characterized CDR3 T-cell lymphomas2. Limit of detection experiments with a subset
and the full set of plasmids result in similar performance – expected level of V-gene
frequency with linearity at low inputs (50-5,000 copies). When plasmids are individually
amplified, then pooled in a background of 100ng leukocyte total RNA, and sequenced
on an Ion 530™ chip, as little as 5 copies of plasmid are detected. To further assess
linearity, pools of plasmids were prepared at various input concentrations ranging 50-5M
copies in a background of 100ng cDNA reverse transcribed from leukocyte total RNA.
Linearity is observed over 5 orders of magnitude. Equimolar pools of plasmids express
plasmid frequencies within one order of magnitude. Sequencing results, number of
productive reads, and number of identified clones are similar when the plasmids are
linearized individually, linearized in bulk, or left intact. Thus, the Oncomine™ TCR-Seq
β assay has made it possible to amplify the full length TCRβ and sequence the human
TCRβ repertoire on the Ion S5.
REFERENCES
1. Dongen et al, Leukemia (2003) 17, 2257-2317
2. Sandberg et al, Leukemia (2007) 21, 230-237
3. Liu et al, PLoS ONE (2016) 11(3): e0152464
ACKNOWLEDGEMENTS
The authors would like to acknowledge the work of all who participated in this program:
Alice Zheng, Alex Pankov, Grace Lui, Gauri Ganpule, Sonny Sovan, Larry Fang, Tyler
Stine, Laura Nucci, Rob Bennett, and Jim Godsey.
Counts
Amplify targets using
Oncomine TCR-Seq β assay
Isolate and quantify RNA
Reverse transcribe
Partially digest amplicons
Ligate adapters
Unbiased output:
AmpliSeqTM V- and
C-region primers
are optimized to
reproduce results
from 600bp 5’-
RACE protocols.
Comprehensive:
400bp read length
offers complete
characterization of
CDR1,2,3.
Figure 2. Sequencing read length
histogram from library generated using
the OncomineTM TCR-Seq β assay run on
an Ion 530 chip. The resul9ng sequencing
libraries are generally 325-350bp in
length. Total read counts are between
15-20M reads, of which 70-80% are
“producJve” (blue+light blue) when run
through our analysis pipeline.
Figure 2. Read Length Histogram
Figure 3. Using 5’-RACE as presumed “truth”
we judge the accuracy of our assay using
correla9on of V-gene usage.
Figure 4. Limit of Detection
Figure 4. Limit of detection/linearity
experiments including 30 plasmid
sequences result in expected level of
plasmid representation (linear with input)
and high sensitivity at low abundance.
a.) Model results show measurements are
linear at low inputs. b.) Experimental
results with 4 plasmids follow the same
pattern as the model data. c.) Libraries
prepared using pooled plasmids at single
known input concentrations (1pg to
0.00001pg = 50,000 to ~5 copies) in a
background of 100ng Leukocyte cDNA.
a.) b.)
c.)
Linear Region
Figure 5. Linearity
a.) b.)
c.)
Figure 3. Correlation to 5’-RACE
b.)
a.)
0.0001
0.0010
0.0100
0.1000
1.0000
0.0001 0.001 0.01 0.1 1 10
Log10ObservedPlasmid
Frequency
Plasmid Input (pg)
K-T1a
HPB-ALL
SUP-T3
SU-DHL-1
Frequency vs Plasmid Input – Individual Plasmids
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