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

[DESCRIPTION]
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. 

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.

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.

This protocol describes methods to estimate: 
A. library fragment size and concentration using both the Bioanalyzer 2100 and Tapestation 4200 workstations.
B. library and pool concentrations using an adaptor-ligating primer-based qPCR assay.

The steps described have been adapted from the technology and methods developed by Agilent Technologies and New England BioLabs (NEB).


This is an open-access protocol distributed under the terms of the Creative Commons Attribution-NonCommercial-ShareAlike.
 







[GUIDELINES]
Storage of Kit Components:

1. Agilent D1000 ScreenTape Assay and Agilent High Sensitivity DNA Assay:
See instructions on packaging.


2. NEBNext Library Quant Kit for Illumina:

All of the individual components of the NEBNext Library Quant Kit for Illumina are stable at –20°C for two years. When not in use, kit components should be stored at –20°C. All components of the kit, including the combined NEBNext Library Quant Master Mix and Primer Mix, are stable for at least 30 freeze/thaws.

Once the Primer Mix has been added to the NEBNext Library Quant Master Mix (see Protocol on page 6 of manual), the resulting mix can be stored at –20°C for seven months. For short-term storage, this mix may be stored at 4°C for up to six weeks.

Reactions can be prepared at room temperature, but unused portions of the kit reagents should be kept on ice after thawing them for use.

[STEPS]
SECTION: Before Starting
1. 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.
SECTION: Assays
2. 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 / 4200

Quantitative PCR of double-stranded sequenceable library molecules:
Adaptor-ligating primer based qPCR assay (NEBNext® Library Quant Kit for Illumina®)
SECTION: Assays
3. 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 Guide

Agilent 2100 Bioanalyzer System (Maintenance and Troubleshooting Guide)

Agilent 2100 Bioanalyzer (Installation and Safety Guide)

Converting ng/µl to nM when calculating dsDNA library concentration
 


If using Illumina Library Prep protocols, refer to the following Library quantification and quality control quick reference guide
3. 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 concentration
 


If using Illumina Library Prep protocols, refer to the following Library quantification and quality control quick reference guide


SECTION: Assays
3. The following reagents are supplied with this product:
  

SECTION: Assays
4. E7630 Product Information and Specifications

Experimental 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 - NEBioCalculator

 


If using Illumina Library Prep protocols, refer to the following Library quantification and quality control quick reference guide