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

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 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.

COVseq is a cost-effective workflow for mass-scale SARS-CoV-2 genomic surveillance

“…COVseq enables near-complete coverage of the SARS-CoV-2 genome. The CUTseq method, which we previously described 21 , enables a cost-effective preparation of highly multiplexed DNA sequencing libraries, by using restriction enzymes to barcode multiple samples before pooling them together into a single library. When the SARS-CoV-2 pandemic started, we sought to adapt CUTseq to sequence many SARS-CoV-2 genomes in parallel at an affordable cost.…”

Sustainability by design: automated nanoscale 2,3,4-trisubstituted quinazoline diversity

“…5A-D). [19][20][21][22] A well-defined pressure pulse on top of a 96-well microliter plate housing the building block stock solutions with holes in the bottom of each well forms a highly precise nanoliter droplet that is released into any target plate (96-, 384-well or higher formats). The I-DOT source plate is located above the target plate that moves underneath.…”

Radiological Correlates of pT3a Kidney Cancer: Importance of Irregular Tumor Sinus Border

“…related to library size due to depth and breadth) rather than sample processing and genomic material extraction, obtaining DNA from multiple regions and mixing them into a single pseudo-bulk would result in minimal additions to the total cost (Supplementary Figure 5a). Given that our direct pooling approach requires no additional reagents (nor modifications to the computational infrastructure), it occupies a flexible middle ground investigators 10 . Furthermore, pooling of tumor regions preserves precious tumor tissue which could be used for further molecular, immunohistochemical, or other profiling, and therefore is a material-efficient alternative to fully unbiased representative sequencing 11 .…”

“…Compared to single region profiling, pooled multi-regional sequencing showed a 12% lower dropout rate (95% CI: 2.0 -22.4%, Welch t-test, p=0.02) and a 13% lower clonality error rate (95% CI: 1.2 -24.9%, Welch t-test, p=0.03) (Supplementary Figure 2b). Reduction in clonality misattribution was robust with the chosen cancer-cell fraction (CCF) threshold, with an estimated Matthew's correlation coefficient (MCC) of 0.73 (with +1 indicating perfect prediction and -1 complete disagreement) (Supplementary figure compared to bona fi de multiregional sequencing and full-scale mixing of left-over tumor tissue 10,11 . We defi ned a metric of cost-eff ectiveness as the change in mutation dropout (or clonality) per tumor relative to the change in cost (Supplementary fi gure 5 b, c).…”