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Bioinformatics Advance Access published online on January 9, 2009
Bioinformatics, doi:10.1093/bioinformatics/btp010
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© The Author (2009). Published by Oxford University Press. All rights reserved. For Permissions, please email: journals.permissions@oxfordjournals.org

Profiling model T cell metagenomes with short reads

René L Warren 1,*, Brad H. Nelson 2 and Robert A. Holt 1

1BC Cancer Agency, Michael Smith Genome Sciences Centre, 675 West 10th Avenue, Vancouver, B.C., Canada, V5Z 1L3
2BC Cancer Agency, Deeley Research Centre, 2410 Lee Ave, Victoria, B.C., Canada, V8R 6V5

*To whom correspondence should be addressed. Mr. Rene Warren, E-mail: rwarren{at}bcgsc.ca


  Abstract

Motivation: T cell receptor (TCR) diversity in peripheral blood has not yet been fully profiled with sequence level resolution. Each T cell clonotype expresses a unique receptor, generated by somatic recombination of TCR genes and the enormous potential for T cell diversity makes repertoire analysis challenging. We developed a sequencing approach and assembly software (immuno-SSAKE or iSSAKE) for profiling T cell metagenomes using short reads from the massively parallel sequencing platforms.

Results: Models of sequence diversity for the TCR beta-chain CDR3 region were built using empirical data and used to simulate, at random, distinct TCR clonotypes at 1-20 parts per million. Using simulated TCRβ (sTCRβ) sequences, we randomly created 20 million 36nt reads having 1%-2% random error, 20 million 42nt or 50nt reads having 1% random error and 20 million 36nt reads with 1% error modeled on real short read data. Reads aligning to the end of known TCR variable (V) genes and having consecutive unmatched bases in the adjacent CDR3 were used to seed iSSAKE de novo assemblies of CDR3. With assembled 36nt reads we detect over 51% and 63% of rare (1 ppm) clonotypes using a random or modeled error distribution, respectively. We detect over 99% of more abundant clonotypes (6 ppm or higher) using either error distribution. Longer reads improve sensitivity, with assembled 42nt and 50nt reads identifying 82.0% and 94.7% of rare 1 ppm clonotypes, respectively. Our approach illustrates the feasibility of complete profiling of the TCR repertoire using new massively parallel short read sequencing technology.

Availability: ftp://ftp.bcgsc.ca/supplementary/iSSAKE.

Contact: rwarren{at}bcgsc.ca

Supplementary Information: Supplementary methods and data available at Bioinformatics online and at ftp://ftp.bcgsc.ca/supplementary/iSSAKE

Associate Editor: Prof. Alfonso Valencia

Received on September 26, 2008; revised on November 28, 2008; accepted on January 1, 2009

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Home page
 Genome ResHome page
J. D. Freeman, R. L. Warren, J. R. Webb, B. H. Nelson, and R. A. Holt
Profiling the T-cell receptor beta-chain repertoire by massively parallel sequencing
Genome Res., October 1, 2009; 19(10): 1817 - 1824.
[Abstract] [Full Text] [PDF]


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