Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice

Abstract

Transposon-mediated forward genetics screening in mice has emerged as a powerful tool for cancer gene discovery. It pinpoints cancer drivers that are difficult to find with other approaches, thus complementing the sequencing-based census of human cancer genes. We describe here a large series of mouse lines for insertional mutagenesis that are compatible with two transposon systems, PiggyBac and Sleeping Beauty, and give guidance on the use of different engineered transposon variants for constitutive or tissue-specific cancer gene discovery screening. We also describe a method for semiquantitative transposon insertion site sequencing (QiSeq). The QiSeq library preparation protocol exploits acoustic DNA fragmentation to reduce bias inherent to widely used restriction–digestion-based approaches for ligation-mediated insertion site amplification. Extensive multiplexing in combination with next-generation sequencing allows affordable ultra-deep transposon insertion site recovery in high-throughput formats within 1 week. Finally, we describe principles of data analysis and interpretation for obtaining insights into cancer gene function and genetic tumor evolution.

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Figure 1: Natural autonomous transposons and nonautonomous two-component systems engineered for insertional mutagenesis in mice.
Figure 2: A versatile platform of mouse models for insertional mutagenesis.
Figure 3: Mechanisms of ATP transposon–mediated mutagenesis.
Figure 4: Library preparation for QiSeq.
Figure 5: DNA fragmentation methods for splinkerette PCR: restriction digestion versus acoustic shearing.
Figure 6: Transposon insertion sites identified by QiSeq.

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Acknowledgements

This work was supported by the Wellcome Trust, the German Cancer Consortium (DKTK), the German Research Council (SFB1243) and the Helmholtz Association (PCCC Alliance).

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Contributions

M.F. and R.R. wrote the manuscript. R.R., A.B., P.L., W.W., L.R., J.C., G.S.V. and A.S. designed or contributed to the development of the transposon systems in mice. M.A.Q., I.B., M.M., R.R., A.S., C.G. and G.S.V. designed or contributed to the development of QiSeq. I.B., M.M., M.F. and A.S. developed the library preparation method for plate formats. R.R., M.F., L.R., J.W., A.P., D.S., T.E. and A.B. provided and analyzed the transposon screening data. H.P., R.R. and G.S.V. developed bioinformatics tools for the analysis of insertional mutagenesis data. J.W. developed/adapted the DNA extraction protocol from formalin-fixed paraffin-embedded tissues for QiSeq.

Corresponding author

Correspondence to Roland Rad.

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The authors declare no competing financial interests.

Integrated supplementary information

Supplementary Figure 1 Read quality score per sequenced base position.

(a) Illumina Q-scores for a normal QiSeq run. Typically, more than 80% of reads reach a quality score higher than 30 across all read positions.(b) DNA extracted from formalin-fixed, paraffin embedded tissue can be sequenced to similarly high quality. (c) Samples with a low transposon mobilisation rate lead to run failure due to loss of focus; most reads consist of identical transposon transgene concatemer sequences instead of random genomic sequence. As a consequence the imaging algorithms fail, resulting in reduced quality scores for a larger fraction of reads or complete drop-outs on single base positions. (d) Spiking the library from (c) with 15% PhiX solves the problem.

Supplementary Figure 2 Mating strategies for conditional transposon screens.

(a) Setup for a tissue specific transposon screen. Transposon mice and conditional transposase mice should be kept separate until the last mating step that generates the experimental cohorts for tumour watch. Cre driver lines should be interbred with the transposon line. For details of the rationales see main text. (b) Similarly, if a sensitizing background mutation is needed, two founder cohorts are generated and maintained as indicated. The final cross produces experimental mice and all combinations of controls.

Supplementary Figure 3 Agilent Bioanalyzer QC traces

(a) Sonicated genomic DNA with a mean fragment length of 250 bp. (b) Genomic DNA after end-repair and A-tailing. Little or no peak shift should be detectable compared to (a). (c) Genomic DNA after adapter-ligation and double SPRI clean-up. Peak shift should be more than 100 bp due to adapter ligation on both ends and fragment size selection towards longer fragments due to double SPRI bead cleanup. (d) Adapter-ligated library after PCR2. Distinct subpeaks indicate amplification of transposon containing fragments over genomic-only fragments.

Supplementary Figure 4 Suggested plate layout for qPCR.

Digits are sample numbers, ‚N‘ denotes no template control, ‚S1‘ through ‚S6‘ denotes KAPA library quantification standards 1-6. Each column of the plate with the diluted library splits into 3 columns on the qPCR plate to obtain quantification in triplicate. Thus, each dilution plate requires 4 qPCR plates.

Supplementary information

Supplementary Figures and Text

Supplementary Figures 1–4, Supplementary Table 1, and the Supplementary Method. (PDF 1228 kb)

Supplementary Note 1

Custom Illumina sequencing recipe. (ZIP 10 kb)

Supplementary Note 2

A copy-and-paste template for simplified ordering of primers and oligos from IDT. (XLS 44 kb)

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Friedrich, M., Rad, L., Bronner, I. et al. Genome-wide transposon screening and quantitative insertion site sequencing for cancer gene discovery in mice. Nat Protoc 12, 289–309 (2017). https://doi.org/10.1038/nprot.2016.164

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