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

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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

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R3

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Size of clones

Clonotype identification

V-gene identification

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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.)ComparisonsofV-geneusageto5’-RACElibraryprepara9onstrategiesgivecorrela9onvaluesrangingfromr=0.90-0.92.b.)Correla9oninV-geneusagebetweenBIOMED-2and5’-RACEarenormallyintheranger~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 receptorheavy 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 withoutexonucleotide 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 cellCDR3 receptors from maternal B and T cell CDR3 receptors in cell freeDNA present in maternal peripheral blood. In this way, fetal B and Tcell health and development may be monitored in a non-invasivemanner.

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 receptorheavy 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 withoutexonucleotide 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 cellCDR3 receptors from maternal B and T cell CDR3 receptors in cell freeDNA present in maternal peripheral blood. In this way, fetal B and Tcell health and development may be monitored in a non-invasivemanner.

Figure 1. Structural differences between fetal and adult B and T cell receptors

FR1 FR2

Diversity(D) Joining(J) Constant(C)

Variablegene(V) CDR3FR3

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

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0.00001 0.0001 0.001 0.01 0.1 1

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Frequency vs Plasmid Input (3) 530 Chips with 10 Individual Plasmids each

50 copies

500 copies

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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.

Cou

nts

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.

Figure2.SequencingreadlengthhistogramfromlibrarygeneratedusingtheOncomineTMTCR-SeqβassayrunonanIon530chip.Theresul9ngsequencinglibrariesaregenerally325-350bpinlength.Totalreadcountsarebetween15-20Mreads,ofwhich70-80%are“producJve”(blue+lightblue)whenrunthroughouranalysispipeline.

Figure 2. Read Length Histogram

Figure 3.Using5’-RACEaspresumed“truth”wejudgetheaccuracyofourassayusingcorrela9onofV-geneusage.

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.)

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K-T1a HPB-ALL SUP-T3 SU-DHL-1

Frequency vs Plasmid Input – Individual Plasmids

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