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Use of synthetic DNA spike-in controls (sequins) for human genome sequencing

Nature Protocols volume 14, pages 2119–2151 (2019)Cite this article

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Abstract

Next-generation sequencing (NGS) has been widely adopted to identify genetic variants and investigate their association with disease. However, the analysis of sequencing data remains challenging because of the complexity of human genetic variation and confounding errors introduced during library preparation, sequencing and analysis. We have developed a set of synthetic DNA spike-ins—termed ‘sequins’ (sequencing spike-ins)—that are directly added to DNA samples before library preparation. Sequins can be used to measure technical biases and to act as internal quantitative and qualitative controls throughout the sequencing workflow. This step-by-step protocol explains the use of sequins for both whole-genome and targeted sequencing of the human genome. This includes instructions regarding the dilution and addition of sequins to human DNA samples, followed by the bioinformatic steps required to separate sequin- and sample-derived sequencing reads and to evaluate the diagnostic performance of the assay. These practical guidelines are accompanied by a broader discussion of the conceptual and statistical principles that underpin the design of sequin standards. This protocol is suitable for users with standard laboratory and bioinformatic experience. The laboratory steps require ~1–4 d and the bioinformatic steps (which can be performed with the provided example data files) take an additional day.

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Fig. 1: Schematic showing the design and use of sequins in NGS experiments.
Fig. 2: Sequin design principles.
Fig. 3: Compatibility of sequins with targeted sequencing.
Fig. 4: Overview of protocol for sequin use in human genome sequencing.
Fig. 5: Calibration of sequin coverage to matched human genome regions.
Fig. 6: Example traces measuring DNA fragment size and abundance.
Fig. 7: Example qPCR assessment of target enrichment for ALK, BRAF, PIK3CA, PTEN and TP53.
Fig. 8: Example of sequin and corresponding human variants.
Fig. 9: Alignment-free comparison of quantitative accuracy between libraries.
Fig. 10: Impact of sequence context on NGS performance.
Fig. 11: Performance evaluation of somatic variant calling by anaquin.
Fig. 12: Comparison of expected human and sequin variants analyzed by targeted sequencing.

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Data availability

All next-generation sequencing libraries and associated data files, including synthetic sequences and variant annotations, are available for download at http://www.sequinstandards.com/resources/#nature_protocols. Please see the ‘Equipment setup’ section and Supplementary Notes 1 and 2 for further details.

Code availability

Anaquin source code is available from https://github.com/sequinstandards/RAnaquin.

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Acknowledgements

The authors would like to thank the following funding sources: Australian National Health and Medical Research Council (NHMRC) Australia Fellowships (1062470 to T.R.M.), APP1108254 (to B.S.K.) and APP1114016 (to J.B). I.W.D is supported by a Cancer Institute NSW Early Career Fellowship (2018/ECF013). T.R.M. and T.W. are supported by a Paramor Family Fellowship. S.A.H. is supported by an Australian Postgraduate Award scholarship. A.L.M.R. is supported by a University of New South Wales Sydney Tuition Fee Scholarship. The contents of the published material are solely the responsibility of the administering institution, a participating institution or individual authors and do not reflect the views of the NHMRC.

Author information

Author notes

  1. These authors contributed equally: J. Blackburn, T. Wong.

Authors and Affiliations

  1. Genomics and Epigenetics Division, Garvan Institute of Medical Research, Sydney, Australia

    James Blackburn, Ted Wong, Bindu Swapna Madala, Chris Barker, Simon A. Hardwick, Andre L. M. Reis, Ira W. Deveson & Tim R. Mercer

  2. St Vincent’s Clinical School, Faculty of Medicine, UNSW Australia, Sydney, Australia

    James Blackburn, Simon A. Hardwick, Andre L. M. Reis, Ira W. Deveson & Tim R. Mercer

  3. Altius Institute for Biomedical Sciences, Seattle, WA, USA

    Tim R. Mercer

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Contributions

J.B., B.S.K. and C.B. contributed materials. J.B. performed the experiments. T.W., I.W.D., S.A.H. and A.L.M.R. carried out the bioinformatic analysis. J.B., T.W., I.W.D. and T.R.M. wrote the manuscript. All authors conceived the study and contributed to manuscript preparation.

Corresponding authors

Correspondence to Ira W. Deveson or Tim R. Mercer.

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Competing interests

The authors declare competing interests: the Garvan Institute of Medical Research has filed patents covering aspects of sequencing controls.

Additional information

Journal peer review information: Nature Protocols thanks Justin Zook and other anonymous reviewer(s) for their contribution to the peer review of this work.

Publisher’s note: Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Related links

Key references using this protocol

Deveson, I. W. et al. Nat. Commun. 10, 1342 (2019): https://doi.org/10.1038/s41467-019-09272-0

Hardwick, S. A., Deveson, I. W. & Mercer, T. R. Nat. Rev. Genet. 18, 473–484 (2017): https://doi.org/10.1038/nrg.2017.44

Deveson, I. W. et al. Nat. Methods 13, 784–791 (2016): https://doi.org/10.1038/nmeth.3957

Integrated supplementary information

Supplementary Figure 1 Example of sequin calibration.

Genome browser views show sequencing alignments within a single sequin standard before (upper) and after (middle) coverage calibration, performed using anaquin ‘calibrate’. During calibration, sequin alignments are down-sampled to achieved matched coverage with the human sample DNA (lower) within sequin regions. This example also shows artifactual enrichment of read-pairs at sequin termini, which occurs during some library preparation methods. Anaquin ‘calibrate’ automatically removes these terminal alignments before calibration. Sequin edge regions (550 bp, by default) are also excluded during the calibration process, as well as downstream anaquin analyses (germline/somatic).

Supplementary information

Supplementary information

Supplementary Figure 1

Reporting Summary

Supplementary Information

Supplementary Notes 1 and 2

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Blackburn, J., Wong, T., Madala, B.S. et al. Use of synthetic DNA spike-in controls (sequins) for human genome sequencing. Nat Protoc 14, 2119–2151 (2019). https://doi.org/10.1038/s41596-019-0175-1

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