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


Session type:

Xiaolu Zhang1,Silvano Garnerone1,Michele Simonetti1,Marcin Nicoś1,Luuk Harbers1,Caterina Marchiò2,Reza Mirzazadeh1,Tiziana Venesio2,Anna Sapino2,Johan Hartman1,Magda Bienko1,Nicola Crosetto1
1Karolinska Institutet,2Candiolo Cancer Institute



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 single library must be produced separately for each sample. Furthermore, library preparation is challenging in the case of low-input 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 validate CUTseq and demonstrate its applicability to both genome and exome sequencing, enabling multi-region genome profiling within single stained FFPE tissue sections, to assess intratumor heterogeneity at high spatial resolution. 


In conclusion, CUTseq is a versatile and cost-effective method for multiplexed DNA sequencing library preparation that can find numerous applications in research and diagnostics.