CUTseq enables multiplexed genome barcoding-amplification for multi-region sequencing of FFPE samples

Formalin-fixed paraffin embedded (FFPE) tissues are widely used in pathological diagnostics.Although DNA extracted from FFPE samples is suitable for massively parallel sequencing, buildingsequencing libraries from multiple FFPE samples remains technically challenging and costly. Here,we present a method, CUTseq, that allows multiplexed genome barcoding, amplification andreduced representation sequencing of DNA extracted from individual FFPE tissue sections ormultiple sub-regions in them. CUTseq uses restriction enzymes to fragment and barcode thegenome at defined locations, and the T7 phage promoter to amplify the DNA sequence surroundingthe cut sites before a sequencing library is prepared. We compared CUTseq with a standard way forpreparing sequencing libraries compatible with Illumina technology using DNA extracted fromFFPE sections of different tumor types, demonstrating a strong correlation between DNA copynumber profiles obtained with two approaches. Furthermore, by applying CUTseq to DNAextracted from dozens of small (<10 mm 2 ) regions inside individual FFPE tissue sections of primarybreast cancers and matched metastases, we were able to study intra-tumor heterogeneity in DNAcopy number, at a spatial resolution so far unattained. CUTseq is a versatile and cost-effectivemethod for multiplexed reduced representation genome sequencing that can be applied to both fixedand non-fixed samples.