Capture and Amplification by Tailing and Switching CATS)

The use of next generation sequencing technologies in the field of epdemiology is still largely unfeasible.

A major problem is the fact that current protocols for deep sequencing library generation require minimal nucleic acids inputs in the order of several tens to hundreds nanograms. Those protocols depend on adapter ligation to the 5’- and/or3’- ends of the target fragments, which is a both material- and time-consuming process. In contrast, the amounts obtainable from many clinical samples that would enable large-scale screening for presence and characteristics of tumor-derived nucleic acids, such as circulating cell-free DNA and RNA in blood plasma, are usually much lower.

In addition, nucleic acids from body fluids and other clinical materials are often very short or highly fragmented, causing considerable difficulties to current fragment library generation approaches. With costs of usually far more than 100 $ per sample, avaiable commerical library generation kits are also still too expensive to enable a regular use in epidemiology studies with thousands of samples.

To address and overcome these issues, we have developed the CATS (Capture and Amplification by Tailing and Switching) protocol that produces ready-to-sequence fragment libraries directly from sub-nanogram amounts of native DNA or RNA in a cost- and time-efficent manner

CATS combines several important advantages compared to existing protocols:

CATS is a ligation-free fragment library generation protocol. Avoiding the need for ligation allows the use extremely low amounts of sample material, down to sub-nanogram range. The material can be used directly and without the need of any prior amplification steps or whole genome amplification before the adapters are attached.

CATS does only take a few hours from sample to library since no particularly time-consumings steps (e.g. ligation or WGA) are involved. The resulting fragment libraries can be directly used for High-Throughput / Next Generation Sequencing or any other fragment library-based analysis.

CATS captures single strands and is naturally strand specific, meaning the directionality of the fragment ends (5‘ and 3‘) is preserved during the capture. While this is especially useful in RNA sequencing, it also enables the detailed study of single-stranded DNA species. Of course, also double-stranded DNA samples can be used, if the strands are denatured first.

CATS is efficient on very short fragments of DNA and RNA, for example non-coding small RNAs or highly fragmented DNA from blood plasma. The lower length limit is determined by the necessities for a minimum informative sequence length during the bioinformatic analysis.

At the same time, CATS also efficiently captures longer DNA/RNA fragments in the sizes used by other library protocols and sequencing technologies, for example artifically fragmented DNA or RNA in the range of 200-300 bp for Illumina technologies.

The consumables costs about $10 per library

The CATS Principle:

CATS principle

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