Friday, 17 October 2014

Indexing 1: A Simple NGS Pooling How-To Guide

Indexes are one of the simplest improvements in the last five years of sequencing, with the most incredible far-reaching effects. Today I will share a complementary pair of posts tackling the problems our customers experience most frequently when submitting indexed libraries for sequencing.

How do I pool my library at a defined concentration?

I get asked this a lot. Our current submission requirements are 10nM - 20nM in 15ul, but what does this mean? Is the total DNA concentration in the pool 10nM, and each individual library therefore much less? Or is it that each library within the pool is at a final concentration of 10nM?

Simply put, our submission guidelines IGNORE your indexes. Quantification and clustering cannot differentiate between indexes on a sample, so all we are interested in is the total quantity of DNA in your pool.  So, for example, if you have five libraries in a pool, the final pool DNA concentration must be at least 10nM - which means that each library within that pool is at least 2nM.

Here is the simplest at-a-glance method to dilute and pool your libraries. For more detailed hints and tips read on to my next post!
  1. Quantify and quality check all of your libraries
  2. Select a goal concentration for pooling - at or below the lowest concentration of your set of libraries.
  3. Make sure this is within our current submission guidelines.
  4. Dilute all of your libraries to that concentration, using Illumina Resuspension Buffer, EB, or 10mM Tris pH 8.5 with 0.1% Tween.
  5. Combine an equal volume of all of your libraries in your pool tube

Ta-da! You are ready to submit your pool for sequencing.

1 comment:

  1. Hi I work in Research and working with some very precious samples from follicular fluid where some of the libraries that we create give us a very poor library molarity. Sometimes as low as 0.5nM. Because of the nature of the research it is essential for us to give the sequencing a go and see what we get. I am however, unsure of how to pool 0.5nM libraries with libraries at 3nM for HiSeq 4000 (taking into account I only have one shot with a limited volume).

    Would be grateful if you can make any suggestions.