CUTseq is a versatile method for preparing multiplexed DNA sequencing libraries from low-input samples

Xiaolu Zhang,Michele Simonetti,Silvano Garnerone,Luuk Harbers,Marcin Nicoś,Reza Mirzazadeh,Tiziana Venesio,Anna Sapino,Nicola Crosetto,Magda Bienko,Caterina Marchiò,Johan Hartman

doi:10.1038/s41467-019-12570-2

Abstract

Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. This procedure is costly and time-consuming, because a library must be generated separately for each sample. Furthermore, library preparation is challenging in the case of fixed samples, such as DNA extracted from formalin-fixed paraffin-embedded (FFPE) tissues. Here we describe CUTseq, a method that uses restriction enzymes and in vitro transcription to barcode and amplify genomic DNA prior to library construction. We thoroughly assess the sensitivity and reproducibility of CUTseq in both cell lines and FFPE samples, and demonstrate an application of CUTseq for multi-region DNA copy number profiling within single FFPE tumor sections, to assess intratumor genetic heterogeneity at high spatial resolution. In conclusion, CUTseq is a versatile and cost-effective method for library preparation for reduced representation genome sequencing, which can find numerous applications in research and diagnostics.

Highlights

Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation
We aimed at developing a versatile method for preparing highly multiplexed DNA sequencing libraries, by barcoding genomic DNA (gDNA) from multiple samples directly after purification
After gDNA is digested, the restricted sites are ligated to specialized double-stranded DNA adapters that contain a sample-specific barcode sequence, a unique molecular identifier (UMI)[9], the RA5 Illumina sequencing adapter, and the T7 promoter sequence

Summary

Introduction

Current multiplexing strategies for massively parallel sequencing of genomic DNA mainly rely on library indexing in the final steps of library preparation. Whole-genome amplification methods, such as DOP-PCR4, MDA5, MALBAC6, and the more recent SCMDA7 and LIANTI8, achieve direct gDNA barcoding during genome amplification, so that multiple samples can be pooled together into a single multiplexed library Such methods are tailored for whole-genome sequencing of single cells, they could, in principle, be used for other applications, for instance to generate multiplexed libraries for multi-region tumor sequencing in tissue sections. Whole-genome amplification methods are very costly, making them hardly applicable to routine diagnostics To overcome these limitations, here we develop a method, which we name CUTseq, that combines restriction endonucleases with in vitro transcription (IVT), to construct highly multiplexed DNA libraries for reduced representation genome sequencing of multiple samples in parallel. We describe a workflow for rapid and costeffective preparation of highly multiplexed CUTseq libraries, which can be applied in the context of high-throughput genetic screens and for cell line authentication

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Conclusion