TITLE: C-SOP-401: Quality Control (QC) of DNA Libraries for Whole Genome Sequencing
AUTHORS: Mihir Kekre

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block"><div class = "justify" style = "text-align:left">The success of fragmentation and size selection is best confirmed using an electrophoretic instrument designed for automated and high throughput use, suitable for applications such as NGS. Unlike gel electrophoresis, the output of electropherograms provides qualitative measurements of 1. median fragment size, 2. distributions of fragments sizes within the sample alongside quantitative estimates like sample concentration. </div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">An ideal electropherogram for bacterial whole genome sequencing (WGS) libraries should reveal a single peak/band of desired size with no tailing and excessive broadening. Prior to the sequencing run, all WGS libraries are checked on a fragment analyser (the most popular of which are Agilent's Bioanalyzer and Tapestation instruments). The assays runs on these instruments are capable of verifying if library size distributions are as expected as well as the unwanted presence of any contaminating adapter-dimers or single-stranded material. These can be a major issue with exclusion-amplification clustering chemistry on Illumina platforms.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">Alongside electropherograms, it is recommended that libraries be quantified to specifically estimate the amount of double-stranded usable library for equal representation within a pool when loaded onto the sequencer. This can be done using fluorometric dye-based quantitation using either a fluorometer (such as Qubit) or real-time quantitative PCR (qPCR). Between fluorometry and qPCR, only the latter can specifically target the adaptor-ligated amplifiable molecules by using primers complementary to the adaptor sequences. Quantifying only these viable sequencing templates gives you the best chance at normalising libraries accurately downstream.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">This protocol describes methods to estimate: </div></div><div class = "text-block"><div class = "justify" style = "text-align:left">A. library fragment size and concentration using both the Bioanalyzer 2100 and Tapestation 4200 workstations.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left">B. library and pool concentrations using an adaptor-ligating primer-based qPCR assay.</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left">The steps described have been adapted from the technology and methods developed by Agilent Technologies and New England BioLabs (NEB).</div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:left"></div></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 class = "text-block"><div class = "justify" style = "text-align:left"><a href="#" style = "text-decoration:underline;color:blue;cursor:pointer;"><span style = ":;"> </span></a></div></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 class = "text-block"><div class = "justify" style = "text-align:left"></div></div><div class = "text-block"><div class = "justify" style = "text-align:justify"></div><style>
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[STEPS]
?. [Before Starting]
Prior to initiating the 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.Create an organised bench space by clearing away all clutter in order to maximize work efficiency and to avoid unnecessary movements that will minimise exposure of sterile materials to airborne and liquid contaminants. Prepare a large bucket of ice to store reagents and samples temporarily during use.
?. [Assays]
Perform the following assays based on the step-wise guides detailed below. Fragment analysis (choose one):- High Sensitivity DNA Assay using Bioanalyzer 2100- D1000 ScreenTape Assay using Tapestation 4150 / 4200Quantitative PCR of double-stranded sequenceable library molecules:Adaptor-ligating primer based qPCR assay (NEBNext® Library Quant Kit for Illumina®)
?. [Assays]
Agilent High Sensitivity DNA Kit Quick Start Guide (Run 2-3 replicates per library sample to obtain a robust mean library fragment size)Agilent High Sensitivity DNA Kit GuideAgilent 2100 Bioanalyzer System (Maintenance and Troubleshooting Guide)Agilent 2100 Bioanalyzer (Installation and Safety Guide)Converting ng/µl to nM when calculating dsDNA library concentrationIf using Illumina Library Prep protocols, refer to the following Library quantification and quality control quick reference guide
Ensure that the mean fragment sizes of the replicates do not vary by >5% of each other.
?. Agilent D1000 ScreenTape Assay Quick Guide for Tapestation Systems (Run 2-3 replicates per library sample to obtain a mean total library fragment size)Agilent 4200 TapeStation System (System Manual)Agilent 4150 TapeStation System (System Manual)Converting ng/µl to nM when calculating dsDNA library concentrationIf using Illumina Library Prep protocols, refer to the following Library quantification and quality control quick reference guide
Ensure that the mean fragment sizes of the replicates do not vary by >5% of each other.
?. [Assays]
The following reagents are supplied with this product:
?. [Assays]
E7630 Product Information and SpecificationsExperimental Considerations - NEBNext Library Quant Kit (E7630)User Manual - NEBNext Library Quant Kit (E7630)Quick Protocol - NEBNext Library Quant Kit (E7630)Detailed Protocol - NEBNext Library Quant Kit (E7630)Data Analysis - NEBNext Library Quant Kit (E7630)Expected Results - NEBNext Library Quant Kit (E7630)Converting Cq values to nM stock library concentrations - NEBioCalculatorIf using Illumina Library Prep protocols, refer to the following Library quantification and quality control quick reference guide
If using a method other than qPCR to quantify your libraries, the subsequent pooling and dilution steps will be according to total molecules rather than sequenceable molecules, and these numbers may be different. This may, in turn, lead to variable representation of fragments on the flowcell. Shorter library molecules cluster more efficiently on an Illumina flowcell, so libraries with longer molecules may be underrepresented in the final counts. Moreover, sequence reads of very low quality are removed by the Illumina sequencer's software, so these clusters are not reflected in the post-filter read counts.If one of the triplicates of a diluted library sample is an outlier (i.e. > 0.5 Cq from the other replicates), then the data from this outlier should be excluded from the data analysis.

For accurate quantitation, it is essential to have at least one library dilution that falls within the range of the DNA Standards 1–4. If all qPCR traces for the diluted library sample fall outside the range of the standards, then the qPCR assay should be repeated with greater (if faster than standard 1) or lower (if slower than standard 4) dilution.

The traces for the NTC may show unexpected amplification due to contamination of library targets, primer-dimers, or amplified products if the qPCR assays have been performed repeatedly. As long as the Cq value for the NTC is far enough away from the dynamic range of the DNA Standards (e.g. Cq of NTC should be > 30), the NTC amplification can be ignored as it will not have any effect on library quantitation. If the NTC shows faster amplification, new NEBNext Library Quant Dilution Buffer should be prepared for the next runs.If one of the triplicates of a diluted library sample is an outlier (i.e. > 0.5 Cq from the other replicates), then the data from this outlier should be excluded from the data analysis.

For accurate quantitation, it is essential to have at least one library dilution that falls within the range of the DNA Standards 1–4. If all qPCR traces for the diluted library sample fall outside the range of the standards, then the qPCR assay should be repeated with greater (if faster than standard 1) or lower (if slower than standard 4) dilution.

The traces for the NTC may show unexpected amplification due to contamination of library targets, primer-dimers, or amplified products if the qPCR assays have been performed repeatedly. As long as the Cq value for the NTC is far enough away from the dynamic range of the DNA Standards (e.g. Cq of NTC should be > 30), the NTC amplification can be ignored as it will not have any effect on library quantitation. If the NTC shows faster amplification, new NEBNext Library Quant Dilution Buffer should be prepared for the next runs.