ThruPLEX Plasma-seq produces highly reproducible NGS libraries from less than 1 ng up to 30 ng of cell-free DNA in plasma.
Starting from less than 1 ng to 30 ng of cell-free DNA, ThruPLEX Plasma-seq generates indexed Illumina NGS libraries that are highly reproducible and consistent in performance between replicates and across different input amounts.
In the first PCR, 6 ng of cell-free plasma DNA was used in a final reaction volume of 20 μL containing 0.25 mmol/L of each dNTP, 3.1 mmol/L MgCl2 (Applied Biosystems, Darmstadt, Germany), 2.0 μL tenfold Buffer II (Applied Biosystems), 0.5 μmol/L of each primer, and 2U AmpliTaq Gold (Applied Biosystems).
We next amplified 2 3 ng of periglomerular cell RNA isolated by the method described above using a MessageAmp II aRNA Amplification Kit (Ambion, USA).
Alu-LTR fragments were amplified from 10 ng of total cell DNA in a 25-µl reaction mixture containing 1× PCR buffer, 3.5 mM MgCl2, 200 µM dNTPs, 300 nM primers, and 0.025 units/µl of Taq polymerase.
The fragments were amplified from 10 ng of total cell DNA in a 25-µl reaction mixture containing 1× PCR buffer, 3.5 mM MgCl2, 200 µM dNTPs, 300 nM primers, and 0.025 units/µl of Taq polymerase.
The detection limit for cccDNA is ∼10 copies cccDNA per reaction (equivalent to 10 ng of total cell lysate DNA).
Previously, the assay's limit for detecting KRAS mutations was determined using 0.1 ng of tumor cell line DNA mixed with 100 ng of wild-type DNA [ 11].
HPV-16 was amplifiable by the TDP3 protocol from 1 ng of SiHa cell DNA when the total reaction DNA was 2 μg (Table 2).
The best protocols were the TDP3 and TDP4 that demonstrated HPV-16 amplification from 0.1 ng of SiHa cell DNA in a total DNA content of 100 ng or 500 ng.
