Combined Genome and Transcriptome (G&T) Sequencing of Single Cells
The simultaneous examination of a single cell’s genome and transcriptome presents scientists with a powerful tool to study genetic variability and its effect on gene expression. In this chapter, we describe the library generation method for combined genom
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ntroduction Recently, a sequencing library-generation method for combined genome and transcriptome sequencing (G&T-seq) on single cells was described by Macaulay et al. [1, 2]. Until then, single-cell sequencing methods had only allowed to either determine individual cellular expression profiles or to examine genomic variation (i.e., copy-number variations or single nucleotide variants). By combining transcriptome and genome amplification methods in one protocol, a tool was created that could correlate genetic variability and its effect on gene expression [1]. We have seen a great need for the implementation of the G&T-seq protocol as part of our automated high-throughput single-cell pipeline at the Wellcome Sanger Institute. The original Nature Protocol written by Macaulay et al. [1] gives a very accurate description of the process and can be implemented by skilled scientists. However, it is a very complex protocol that is hard to scale to high sample numbers and is also sensitive to experimental errors. During the implementation
Valentina Proserpio (ed.), Single Cell Methods: Sequencing and Proteomics, Methods in Molecular Biology, vol. 1979, https://doi.org/10.1007/978-1-4939-9240-9_20, © Springer Science+Business Media, LLC, part of Springer Nature 2019
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into our high-throughput pipeline, we have identified potential pitfalls, which might not be obvious to less-experienced users, and improved on the robustness of this protocol to allow for its routine use in high-throughput genomics laboratories. Before endeavoring into the G&T library preparation method, one should understand that the scientific question will have a great impact on the cost per sample and the complexity of the experiment. The G&T method itself provides a robust separation of cellular nucleic acids into distinct single-cell RNA and DNA samples that can be subjected to a range of library preparation methods from these materials. For the RNA, we recommend preparing fulllength cDNA samples to maximize insights; however, 30 or 50 enrichment methods will also yield valuable gene expression data. For the DNA, the operator must make a decision that fundamentally impacts on the analysis that is available later on. For single-cell single-nucleotide variant (SNV) analysis of genomic DNA, a phi29-based multiple displacement amplification (MDA) method is the best choice since phi29 has been shown to have very high DNA amplification accuracy [3]. To get accurate SNV data from amplified DNA products, we suggest using a subsequent no-PCR library preparation method and sequencing of the individual cells to sufficient coverage (e.g., 10–30) to accurately determine SNVs in lower coverage regions. At the time of writing, sequencing individual cells to 10 coverage is still expensive. We therefore suggest to perform an up-front genotyping assay on the individually amplified DNA samples to assess the coverage of the amplified single-cell product and enable cherrypicking of a high-quality subset of samples for library
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