High-throughput DNA sequencing identifies novel CtIP (RBBP8) variants in muscle-invasive bladder cancer patients

Sarah J. Jevons, Angela Green, Gerton Lunter, Christiana Kartsonaki, David Buck, Paolo Piazza, Anne E. Kiltie*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

1 Citation (Scopus)


Background: Germline mutations in DNA damage signalling and repair genes predispose individuals to cancer. Rare germline variants may also increase cancer risk and be predictive of outcomes following cancer treatments, but require high-throughput sequencing (HTS) for detection in large cohorts. Objective: To use a dual indexing system on a HTS platform to detect novel variants in CtIP (RBBP8) which may be associated with clinical outcomes following radiotherapy treatment for bladder cancer. Methods: All exons and flanking introns of CtIP were amplified from germline DNA from bladder cancer patients using seven primer pairs by automated long-range PCR. Amplicons were pooled, fragmented and ligated to adaptor sequences. One of 96 tag sequences was introduced at each end by PCR. Sequencing was performed on a single flow cell of an Illumina MiSeq. Reads were mapped by Stampy and variants called by Platypus. For phasing experiments, target regions were amplified and cloned for Sanger sequencing. Results: Of 201 samples, 160 were successfully amplified. Eleven CtIP variants were called, within the exons and 15 bp adjacent intronic DNA, including eight known variants from the 1000 Genomes project, plus three previously unreported variants now confirmed by Sanger sequencing. In two individuals, phasing experiments showed two variants of interest to be on separate alleles, likely to result in stronger impairment of gene function. Conclusions: We have demonstrated proof of principle for dual indexing on 160 samples on one MiSeq flow cell sequencing surface, and show that for the CtIP gene multiplexing of up to 720 samples would provide sufficient coverage to achieve >98% detection power for rare germline variation, reducing HTS costs substantially.

Original languageEnglish
Pages (from-to)31-44
Number of pages14
JournalBladder Cancer
Issue number1
Publication statusPublished - 2015

Bibliographical note

Funding Information:
This project was funded by the Nuffield Department of Clinical Medicine/University of Oxford Research Fund for Proof of Principle High Throughput Sequencing Experiments, with High-Throughput Sequencing data generated by the High-Throughput Genomics groups at the Oxford Genomics Centre, Wellcome Trust Centre for Human Genetics funded by the Wellcome Trust grant reference 090532/Z/09/Z and MRC Hub grant G0900747 91070. SJ was funded by an MRC studentship awarded to the Department of Oncology, AK was funded by CRUK programme grant C5255/A15935.

Publisher Copyright:
© 2015 – IOS Press and the authors. All rights reserved


  • Biomarkers
  • Bladder cancer
  • CtIP
  • Dual indexing
  • Next generation sequencing
  • Radiotherapy
  • RBBP8


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