Tuesday 17 June 2014

Agilent tools to help with your NGS pooling

Pooling samples for multiplexed sequencing has become the norm for many researchers, especially in the light of 1TB sequencing in 6 days! However many users struggle to get pools nicely balanced. I've just seen a tool I think is helpful so I thought I'd share it with you.

Agilent already provide the Bioanalyser which hopefully anyone who's pooling will already be aware of and will be using to accurately determine total fragment length (not just insert). You'll need this length to get the best out of a replicated KAPA Illumina library quant qPCR. After these steps you can calculate the nM concentration of each library, but then you still need to pool them, which most protocols would handle with a two-step normalisation and pooling.

Agilent provides a better solution in their QXT capture manual "Featuring Transposase-Based Library Prep Technology". Simply use this to combine libraries so each indexed sample is present at equimolar concentration, by pooling volumes as determined by the calculation. They provide an example table for multiplexing of four libraries (see below). The neat feature of their method over those I've used or seen elsewehere is that normalisation and pooling are achieved in a single step. Enjoy!


  1. I've used this method before. One massive advantage is that you can normalise up if you have a few low concentration samples (as long as you have sufficient higher concentration samples to balance the total volume). Just add a larger amount for the those low samples and remove any excess volume from the amount of low TE buffer you need to add.
    The downside to this method is you need to start with samples roughly the same (as in the example above). Large variations in starting concentrations leads to having to pipette either large or small volumes.

  2. Large variations might also suggest that the starting samples were not the same quality or quantity as each other or something happened during library-prep. This itself can be a useful QC.

  3. I can not understand how can you set the final desired volume of the pool and use this value for each library, since the final volume will depend on the other libraries in the pool. Of course, if you end up with less than the desired volume, TE can be used, but using this method, your total volume will probably be higher than the desired.

    1. I'm using an excel sheet for calculate this.

  4. I had a few questions to ask about the pooling of libraries for agilent sureselect assay. I working with 3 samples, based on my bioanalyzer results ,my Index tagged DNA Concentration is (sample1): 76nM, (sample2):76nM and (sample2): 79nM.

    So, with this formula above for sample 1

    v (f)=20

    Volume to use (sample 1) =0.87

    Volume to use (sample 2)=0.87

    Volume to use (sample 3)=0.84

    Please can anyone tell me if this is correct. I will be sequencing it on Nextseq 500(Midoutput kit)150 cycles with a seeding concentration of 1.3pM

    1. Stick the formula into Excel if you prefer. Your libraries are almost equivalent so there is no need to use this formula on this occasion. Simply mix them at 3ul each and add 13.8 of low TE to get approximately a 10nM stock. We'd always check this stock with qPCR before clustering.

  5. Hello,

    I had a question regarding the Post capture index tagged Library concentration for SureSelect XT? .Does it have to be within the range of 10nm-25nm? The example above shows C(i) within that range.

    Is it ok if concentration of Index Tagged library is High (70-100nm). Can we dilute and Pool it to 10nm. Will it affect capture performance and sequencing?



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