We have been running Nextera in my lab for a while now and had some great success e.g. 600x C. elegans genomes. With the release of Illumina's rapid exome kits Nextera is proving to be a versatile and useful addition to our lab tools. Unfortunately it has at least one significant weakness; the need to accurately quantify dilute DNA.
Nextera requires a precise ratio of DNA to transpososomes. Get this wrong and the libraries may not be great and might be unsequenceable.
The standard prep calls for Genomic DNA at 2.5ng/μl and the Nextera XT wants just 0.2ng/μl. We find that many users struggle to get this right for their samples. Most see an improvement if they use a good quantifiaction method and Illumina provides recommendations in the manuals. The use of a double-stranded DNA intercalating dye (we use QuBit) means single-stranded DNA, RNA and other contaminants don't confuse the readings. Nanodrop is a no no for Nextera!
Using SPRI beads to improve the protocols: I have been thinking how we might use a SPRI bead step at the start of a project to capture a very specific amount of DNA. Illumina are using SPRI for bead-based normalisation at the end of the process, so why not add it at the start?
The method is simple enough and would be much quicker for labs without fluorimetric plate readers who have to QT each sample one at a time.
We've not started testing a method yet but next time we're running a Nextera protocol we'll be giving it a go.
As far as I'm aware, such beads are adequate for library normalisation as the targets are relatively small. I'm not sure how these beads would perform on large strands of DNA, such as found in a genomic DNA extraction or even a LR-PCR.
ReplyDeleteI tried adjusting the bead concentration as well as the NaCl concentration to see if limiting one of those reagents will allow normalization to occur. I found no matter what, my recovery is proportional to the amount of beads and the starting mass of DNA. Binding efficiency drops off once NaCl falls below 1M (in bead solution). Wondering if a blocking agent is the key to making this work? I am jealous of the bead-based normalization found in Nextera and TSCA protocols and have found both to work splendidly. As it is now, if I am doing an amplicon prep, I have to pico quant everything (plate reader, but still takes time and costs reagent and plates), then train the robot to pool everything. Quite time consuming and costly.
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