Lifeng Lin, Tim Looney, Geoffrey M. Lowman, Denise Topacio-Hall, Jian-ping Zheng, Elizabeth Linch, Lauren Miller, Mark Andersen and Fiona Hyland, Thermo Fisher Scientific, South San Francisco, CA, USA, 94080

RESULTS

Using 5’-RACE as presumed “truth” we judge the accuracy of our assay using correla9on of V-gene usage.

A) Comparisons of V-gene usage to 5’-RACE library prepara9on strategies give correla9on values ranging

from r = 0.90-0.92; Correla9on in V-gene usage between BIOMED-2 and 5’-RACE are normally in the

range r ~ 0.75-0.80

B) 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. 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.

C) Count of input T cells vs detected clones.

Figure 5. Identification of novel polymorphismsINTRODUCTION

TCRβ immune repertoire analysis by next-generation sequencing is emerging as a

valuable tool for research studies of the tumor microenvironment and potential immune

responses to cancer immunotherapy. Generation of insight from immune repertoire

profiling often requires comparative analysis of immune repertoires across research

sample cohorts representing immune responses to defined antigens or

immunomodulatory agents. Here we describe the development of a computational

framework enabling large-scale comparative analysis of immune repertoire data on

cloud-based infrastructure.

TCRβ receptors were amplified from matched peripheral blood and tumor biopsy cDNA

using AmpliSeqTM multiplex primers targeting the Variable gene Framework 1 and

Constant gene to produce an amplicon spanning all three CDR domains. To evaluate

assay performance, we sequenced TCRβ rearrangements from donor peripheral blood

leukocyte (PBL) cDNA that had been spiked with 30 reference rearrangements taken

from literature. Raw data was uploaded to the Ion Reporter data analysis platform for

clonotype annotation and storage to enable rapid downstream comparative analysis of

repertoire features.

We demonstrate the ability to rapidly compare clonotype data across sample cohorts

and find that a subset of clones identified in peripheral blood are also found in matched

tumor samples. Peripheral blood-derived repertoires typically contained 10-100 fold

more distinct clones than found in tumor, with correspondingly higher estimates of

diversity via the Shannon Index. Results from sequencing of spike-in reference

rearrangements indicate that the assay is accurate and sensitive over 5 logs of input

template amount while showing minimal amplification bias. Technical replicates showed

high concordance (r >.96) in the frequency of detected clones, indicating that results

were reproducible and samples were sequenced to an appropriate depth. Comparison

of AmpliSeqTM multiplex PCR-derived data to that produced by 5’ RACE or BIOMED-2

primers revealed the AmpliSeqTM solution to provide comprehensive and unbiased

coverage of the human TCRβ repertoire.

MATERIALS AND METHODSIon AmpliSeqTM primers are designed based on sequences downloaded from IMGT (1)

(http://www.imgt.org/vquest/refseqh.html). Forward primer set was designed to target

the FR1 region of all TRBV loci; reverse primer set was designed from the two TRBC

loci. The resulting amplicon spans across all CDR regions of the mature mRNA

molecule. Every known variable gene allele have at least one perfect matching forward

primer.

TCRB sequences are amplified using non-FFPE RNA from tumor biopsy, peripheral

blood or sorted cells, followed by multiplex sequencing via the Ion S5 530 chip (15-20M

reads). PCR and sequencing errors are eliminated before clone reporting. In some

cases, an individual will possess a plurality of clones that do not match any IMGT

variable gene allele; this may indicate presence of a novel allele. If sufficient clone

support exists, Ion Reporter classifies the sequence as a putative novel variant. As a

last step, putative variants are compared to those found in the Lym1k database (2)

derived from 1000 genomes data.

CONCLUSIONSWe have developed a computational framework to enable rapid analysis of large immune

repertoire datasets derived from AmpliSeq-based sequencing of human TCRβ receptors via

the Ion Torrent S5. The AmpliSeqTM procedure, which features the ability to produce

uniform and reliable results in extremely highly multiplexed PCR, is well suited for immune

repertoire sequencing applications.

REFERENCES1. Lefranc et al. Nucleic Acids Res (2015). 43:D413

2. Yu et al. J Immunol (2017) 198:2202

3. Ye et al. Nucleic Acids Res (2013) 41:W34

ACKNOWLEDGEMENTSThe authors would like to acknowledge the work of all who participated in this program:

Alex Pankov, Grace Lui, Gauri Ganpule, Sonny Sovan, Xinzhan Peng, Larry Fang, Tyler

Stine, Laura Nucci, Rob Bennett, and Jim Godsey.

A computational framework for large-scale analysis of TCRβ immune repertoire sequencing data on cloud-based infrastructure

Thermo Fisher Scientific • 200 Oyster Point Blvd • South San Francisco, CA 94080 • www.lifetechnologies.com

Figure 4 Comparative sequencing

Figure 2. Analysis workflow of Ion Reporter

Annotate the V, D and J gene

for each rearrangement by

comparing to IMGT database

FR1-C multiplex PCR

Report clones and secondary

repertoire features

Report novel alleles

Ion Reporter Workflow

Eliminate PCR and

sequencing errors

Figure 3. Performance Benchmarking

Figure 1. TCRβ AmpliSeq primer locations

Polymorphism within the TCRB variable gene (TRBV) has been linked to chronic autoimmune

diseases. Existing sequencing assays targeting CDR3 region risks missing imporantant

polymorphsims in the CDR1 and CDR2 regions. The AmpliSeq-based sequencing assay

generates sequences of all three CDRs, making the detection of previously unknown

polymorphisms possible.

Example of a non-synonymous IMGT variant. IgBLAST (3) alignment of an allele having two amino acid

substitutions compared to the best matching IMGT allele. This particular allele was detected in our sample

cohort and the Lym1K database derived from 1000 genomes data.

A collection of tools specifically designed to analyze

immune repertoire data has been built into a preset

workflow in the Ion Reporter platform. The V, D and J

gene portions of the reads are identified by comparing

against the IMGT database and the subtypes assigned

accordingly. The frequency of each V-gene type and

clonotype are summarized and ranked in a report table

(D).

Visualizations are generated based on the clonotype

data, including bar-plots of allele frequency (A), heat-

map of V-J pairs (B) and a set of interactive

spectratyping plots by evenness, Shannon-diversity,

largest-clone frequency and number of clones in each

cluster (C).

A

C

C

A

B

B

D

T-cell receptor repertoire generated from different samples

can be analyzed as a cohort using the “Compare Samples”

function in Ion Reporter. A summary table showing the count

of each CDR3 region and its frequency in all samples is

generated. (above)

Questions such as number of shared clones across samples

or different sequencing runs can be easily extrapolated. We

have used this analysis to compare T-cell repertoire between

tumor infiltrating leukocytes and leukocytes drawn from

peripheral blood, identifying sharing clones between the two

sample types, as well as clones unique to tumor. (left) TRADEMARKS/LICENSING For Research Use Only. Not for use in diagnostic

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