High quality NGS library prep is enhancing understanding of intra-tissue heterogeneity

The final step to achieve parallel sequencing of genomic DNA is relying on library indexing. This crucial step can bear some difficulties, as it is time-consuming and expensive. Further DNA, that is extracted from FFPE (formalin-fixed paraffin-embedded) tissue blocks is often not intact, which would be required for whole genome amplification.

However, vast collections of FFPE samples still constitute a cornerstone of pathology and the information contained in these samples is crucial to understand or example the development of cancer. While parallelly, the methods to gain proteomic data of multiple regions of FFPE samples are improving, also the genomic sequencing of DNA has made huge steps towards covering multiple regions within the same tumor samples, it is however still limited to covering large regions. This, together with the still relatively high costs limits the applicability of multi-region tumor sequencing to routine cancer diagnostics.

The group around Nicola Crosetto, has developed an exciting new method that has a lot of potential to overcome the above-described difficulties. The method termed CUTseq uses restriction endonucleases and in-vitro transcription (IVT), to generate highly multiplexed DNA libraries, from both cell and low-input FFPE samples. Thus, they were able to profile DNA copy number levels in multiple small regions of individual FFPE tumor sections.

The I.DOT was a used by the researcher to carry out several of the subsequent dispensing steps, starting from low amounts of DNA, followed by the restriction enzymes/buffers and subsequently the CUTseq adapters, which were differently barcoded and numerous more reagents, which can be found in the Supplemental of the original publication.

Finally, the researchers’ workflow was able to profile DNA copy number levels in multiple small regions of individual FFPE tumor sections.

Thank you for your ground-breaking work, Xiaolu Zhangand Nicola Crosetto. CUTseq is a versatile and cost-effective method for library preparation which really will find numerous applications in research and diagnostics.

View the cost of reagents and number of samples per reagent.

Source: https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6802095/

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