Wednesday 23 September 2015

Rubicon Genomics webinar on making cfDNA/ctDNA NGS libraries

Rubicon Genomics have a webinar on Creating Cell-free DNA Libraries with ThruPLEX Plasma-seq that was broadcast on September 17th 2015. The new PlasmaSeq kit has a very simple, 3-step, 15 minutes hands on time, single-tube workflow.

Dr. Kamran Shazand, Rubicon Genomics' Director of Applications presented an intro to cell-free DNA - generally 170bp fragmented DNA, which is the result of degradation by apoptocic enzymes, and that can appear in dimeric and trimeric nucleosomal fragments. Rubicon's earlier chemistry ThruPLEX was use in 2012/12 to sequence foetal and tumour genomes and it has a recommended input of 1-30ng of DNA.

The new PlasmaSeq kit seems to make very nice libraries. Internal data from Rubicon showed better performance against NEBnext and KAPA Hyper, with high diversity, low PCR duplication rates and good GC coverage. Most interestingly Rubicon have built a target capture pipeline using their PlasmaSeq kits, Agilent SureSelect and IDT blockers allowing exome sequencing from as little as 1ng of input DNA.

Methods: optimised DNA repair and ligation of stem-loop adapters, PCR to extend, cleave and amplify the library. You should end up with a major peak at 310bp for cfDNA libraries.

Rubicon also talked about their work looking into how molecular indexes night be added to allow removal of PCR duplicates to PlasmaSeq - -send them your comments if you are interested.

Disclosure: I am a user of Rubicon library prep technology; but also Illumina, NEB, Enzymatics, and many others too! I am an author on the ctDNA paper from 2012 mentioned above and in their webinar. I am on the SAB of Rubicon.

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