Saturday 16 July 2016

Whole genome amplification improved

A new genome amplification technology from Expedeon/Sygnis: TruePrime looks like it might work great for single-cell and low-input anlyses - particularly copy number. TruePrimer is a primer-free multiple displacement amplification technology. It uses the well established phi29 DNA polymerase and a new TthPrimPol primase, which eliminates the need to use random primers and therefore avoids their inherent amplification bias. The senior author on the TthPrimPol primase paper, Prof Luis Blanco, is leading the TruePrime research team.

I saw a recent poster which had results demonstrating equal amplification and homogenous coverage (see image above), no primer artefacts, and high identification of both SNPs and CNVs. TruPrime gave very similar CNV data to unamplified DNA with very little apparent amplification or coverage bias from low coverage whole genome sequencing (12 million reads). Competitors "R" and "G" did not look so good.

What does TthPrimPol do in the cell: TthPrimPol is a DNA and RNA primase with DNA-dependent DNA and RNA polymerase activity. It is a unique human enzyme capable of de novo DNA synthesis solely with dNTPs and is found primarily in the nucleus - TthPrimPol -/- cells show inefficient mtDNA replication, but it is not an essential protein. In the mitochondria TthPrimPol provides the primers for leading-strand mtDNA synthesis in the replication fork. It is an important protein in the mitochondria where the highly oxidative environment leads to replication stress and and genome instability. It is also capable of reading through template lesions such as 8oxoG, a common DNA lesions produced by reactive oxygen species that causes G to T and C to A substitutions. This may have auseful application in the amplification of FFPE damaged DNA.

Using TruPrime in single-cell sequencing: I can see several opportunities for using this technology in my lab, including both single-cell systems: 10X Genomics and Fluidigm C1 for future copy-number methods. It is also likely to be useful for other low-input experiments and we're likely to couple it with Nextera XT or similar.

I'm sure we'll see some great work using this enzyme if it really works as well as the company suggest - if you are using TruPrimer please do let me know how you are getting on!

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