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

2019 unpublished
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
more » ... estriction 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. 4. Pool the volume in multiple wells into one 1.5 ml LoBind tube Note: in order to be pooled together, multiple wells must have different sample barcodes. The number of wells pooled together depends on the total number of samples, and on the desired complexity of the final library. In general, libraries containing many samples will need to be sequenced deeply in order for each sample to receive a sufficient number of reads. In our experience, we have successfully sequenced libraries containing up to 96 different barcodes in a single NextSeq 500 run, obtaining a number of reads per sample sufficient to perform reproducible DNA copy number profiling (see Figure 3 ). DAY 5 (DAY 3 for high-throughput CUTseq) DNA cleanup Note: at this point, the same procedure is followed for single-sample and multiplexed libraries. Below, we describe a standard DNA precipitation cleanup procedure. However, silica-based columns or AMPure XP beads can also be used, according to the manufacturer's instructions.
doi:10.21203/rs.2.1742/v2 fatcat:boli4ojwffahhh5vqpxwsnbbui