TITLE: C-SOP-501: Normalisation and Pooling of DNA Libraries for Illumina Whole Genome Sequencing
AUTHORS: Mihir Kekre

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Normalisation in next-generation sequencing (NGS) is the process of equalising the concentration of multiple DNA libraries for the purpose of multiplexing. Multiplexing helps maximize the use of expensive NGS technology, enabling  parallel sequencing of hundreds, to often thousands of libraries on a single flowcell, thereby driving down per sample costs.</div><div class = "text-block">Uneven library concentrations from different types and qualities of samples can lead to inconsistencies in data quality. Libraries with a high concentration are likely to be overrepresented on the flowcell while those with low concentration are underrepresented. Overrepresentation isn’t necessarily a problem, likely increasing read depth, though it does waste the run's finite data capacity. Underrepresentation might result in poor read depth and unreliable data, wasting capacity and potentially precious library material. From a cost standpoint, wasted capacity means means additional work time re-preparing libraries, time that could be better spent on downstream analysis or preparing the next batch of libraries. From an application and outcome standpoint, analyses and decisions based on potentially inaccurate or incomplete data will at best confuse research results or lead to repetition of experiments and at worst lead to clinicians missing key information that could assist in a more appropriate treatment avenue.</div><div class = "text-block">Normalisation addresses these challenges ensuring every library is represented equally and sequenced to sufficient depth.</div><div class = "text-block">Prior to normalisation, there are several options for quantitating library preps, varying in ease and accuracy.</div><div class = "text-block"><ul style = "list-style-type:disc;"><li style = "counter-reset:ol0;list-style-type:disc;">The quickest and most convenient methods (i.e. spectrophotometry-based) tend not to be that accurate.</li><li style = "counter-reset:ol0;list-style-type:disc;"></li><li style = "counter-reset:ol0;list-style-type:disc;">The most accurate methods, like quantitative PCR (qPCR), take time and precision, and rely on knowing the average fragment size in each library for dilution calculations.</li></ul></div><div class = "text-block"><ul style = "list-style-type:disc;"></ul></div><div class = "text-block"><span>One crucial factor influencing the accuracy of quantitation and subsequent normalisation is whether the quantitation method can specifically count adaptor-ligated (i.e. amplifiable) double-stranded DNA (dsDNA) molecules. These are the only molecules that will cluster on the flowcell and contribute to sequencing output. </span><a href="http://emea.support.illumina.com/bulletins/2017/03/best-practices-for-manually-normalizing-library-concentrations.html" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Illumina’s best practice</span></a><span> suggests using fluorometric or qPCR-based quantitation with most types of genomic DNA libraries.</span></div><div class = "text-block">It’s interesting that no single method provides all the data you need with enough accuracy for normalisation. Though fluorometry and qPCR enable the most accurate quantitation, neither can estimate average fragment size. So, it’s often still necessary to check this by electrophoretic analysis (i.e. fragment analysers).</div><div class = "text-block">However, between fluorometry and qPCR, only the latter can specifically target useful adaptor-ligated molecules by using primers complementary to the adaptor sequences. Quantifying only these viable sequencing templates gives you the best chance at normalising your libraries accurately. Adaptor ligation efficiency can vary between individual samples and batches. It’s reliant on enzymatic reactions that could be affected by impurities and differences in the quality of starting material. So, quantitating with no specificity for adaptor-ligated molecules (fluorometry) means you’re more likely to overestimate the sequencing-competent library concentration and over-dilute. Fluorometry (being cheaper and less accurate) can only substitute qPCR when the starting material and the end repair/adaptor ligation step of your library prep workflow are of high and consistent quality.</div><div class = "text-block"><span>This is an open-access protocol distributed under the terms of the </span><a href="https://creativecommons.org/licenses/by-nc-sa/4.0/legalcode" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;">Creative Commons Attribution-NonCommercial-ShareAlike</span></a><span style = ":;">.</span></div><div style = "text-align :; float : ;"><img style = "" src = "https://protocols-files.s3.amazonaws.com/files/cuzrbf8zp.png?X-Amz-Algorithm=AWS4-HMAC-SHA256&X-Amz-Credential=AKIAIWSNCI5SNCPTWTQQ%2F20210112%2Fus-east-1%2Fs3%2Faws4_request&X-Amz-Date=20210112T173149Z&X-Amz-Expires=604800&X-Amz-SignedHeaders=host&X-Amz-Signature=1b2f1a454c966fddb1af93dd8213eb2cd4cbeb804c6ceee4a5e1185f39d2f243" /></div></div>

[STEPS]
?. [Before Starting]
Prior to initiating this protocol, ensure that all active workbenches are cleaned with 80% ethanol, all relevant personal protective clothing is worn and the work area is prepared according to local GLP guidelines for molecular methods.
?. [Before Starting]
Ensure that you have all of the following library QC metrics obtained using the recommended quantification methods described in 'C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing'.1. Mean total library fragment size (in bp) - output from the Bioanalyzer 2100 or Tapestation 4150/4200.2. Mean library concentration (in nM) - output from a qPCR quantification assay.
If your final mean library concentration is represented in ng/µl, convert this value to nM using the following link:Converting ng/µl to nM When Calculating dsDNA Library Concentration
?. [Library Normalisation]
Concentration normalisation is achieved by diluting an aliquot of the stock library in a diluent (nuclease-free water or 10 mM Tris-HCl pH 8.5).
For most Illumina sequencing platforms, 2–4 nM for each library is the preferred final concentration prior to denaturation and final loading steps; consult the respective instrument user guides for more information.
?. [Library Pool Creation (for Multiplexed Libraries)]
Volumetric pooling: Combine equal volumes of each normalised library in a 2.0 mL microcentrifuge tube.
?. [Library Normalisation]
Calculate the dilution volumes for each library using the following equation:(C1)*(V1) = (C2)*(V2)where, C1 = concentration of stock library (nM)V1 = volume of stock library that is to be diluted (µl)C2 = desired concentration of the final solution (nM)
For the most even representation of libraries in the subsequent pooling step and the most reliable cluster density on the Illumina instrument, ensure that all pipetted volumes are preferably not less than 4µl (or at the very least 2 µl). Pipetting less than 2 µl can introduce significant errors in final library pool concentration. Therefore, adjust calculations to make sure that appropriate volumes are used. For highly concentrated libraries, use one or more intermediate dilutions as shown below.For eg. the following calculations demonstrate how to dilute 3 libraries (with different starting concentrations of 15 nM, 20 nM, and 50 nM) to a final concentration of 4 nM in a final volume of 15 µl for each library.
?. [Library Normalisation]
Mix the calculated volumes of library and diluent together to obtain a normalised library solution for each sample.
?. [Library Pool Confirmatory QC]
Quantify the library pool using a robust qPCR assay, like the one described in 'C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing' (NEBNext® Library Quant Kit for Illumina®) and compute the pool concentration in nM.
i. Provided the dilution and pooling was done accurately, the final pool concentration should match the expected value chosen between 2-4 nM (step 3-4).ii. Under extreme conditions of budget limitations or reagent shortages, step 9 could be skipped. However, be advised that downstream results like clustering densities and sequencing run performance may vary greatly.
?. [Additional Information & Troubleshooting]
Normalisation and Pooling CalculatorLibrary quantification and quality control quick reference guideConverting ng/µl to nM when calculating dsDNA library concentrationFor library denaturation and final loading dilution instructions:iSeq™ 100 System GuideMiniSeq™ System Denature and Dilute Libraries GuideMiSeq™ System Denature and Dilute Libraries GuideNextSeq™ 500/550 System Denature and Dilute Libraries GuideNextSeq 1000/2000 Sequencing System GuideHiSeq™ Systems Denature and Dilute Libraries Guide               -  cBot™ System Guide for denaturing and diluting libraries for the HiSeq 3000/4000 NovaSeq™ System Guide
?. [Library Pool Creation (for Multiplexed Libraries)]
Gently pipette the contents up and down 15 times to mix thoroughly. Alternatively, vortexing is also allowed.
?. [Library Pool Creation (for Multiplexed Libraries)]
?. [Library Pool Confirmatory QC]
The normalised pool is now ready to be denatured and sequenced. For detailed instructions on denaturation procedures, use the appropriate instrument denaturation and dilution guide provided under Additional Information & Troubleshooting.