Nov 25, 2022
Library clean up and quality control for Illumina sequencing
- Katherine Smollett1,
- Lily Tong1,
- Kyriaki Nomikou1,
- Jenna Nichols1,
- Kirsty Kwok1,
- Ma. Jowina Galarion1,
- Daniel Mair1,
- Ana Filipe1
- 1MRC-University of Glasgow Centre for Virus Research
Protocol Citation: Katherine Smollett, Lily Tong, Kyriaki Nomikou, Jenna Nichols, Kirsty Kwok, Ma. Jowina Galarion, Daniel Mair, Ana Filipe 2022. Library clean up and quality control for Illumina sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.n2bvj861ngk5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 26, 2022
Last Modified: November 25, 2022
Protocol Integer ID: 71839
Keywords: Next generation sequencing, Illumina, Ampure clean up, Daisy chains, Adapter dimer removal
Disclaimer
Abstract
There are many different ways of preparing libraries for Illumina sequencing but when it comes to the post-library preparation clean up and quality control (QC) there are common rules to apply. For reliable sequencing results it is essential that certain criteria are reached and so accurate QC is crucial. Here we describe a standard library clean up and QC with tips for how to rectify common problems including low yield, adapter dimers and daisy chains.
Materials
Reagents:
Agencourt AmPure XP beadsCatalog #A63880
KAPA HiFi HotStart Library Amplification Kit Illumina® PlatformsRocheCatalog #KK2620
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
High Sensitivity D5000 ReagentsAgilent TechnologiesCatalog #5067-5593
Additional reagents:
10 mM Tris pH8
Nuclease-free water
Absolute Ethanol
QC Equipment:
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
Protocol materials
Agencourt AmPure XP beadsCatalog #A63880
In Materials and 2 steps
KAPA HiFi HotStart Library Amplification Kit Illumina® PlatformsRocheCatalog #KK2620
Materials, Step 45
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Materials, Step 21
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
Materials, Step 34
High Sensitivity D5000 ReagentsAgilent TechnologiesCatalog #5067-5593
Materials, Step 34
Safety warnings
Qubit reagent is known to bind nucleic acid and is provided as a solution in DMSO. Treat the Qubit dsDNA HS Reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations.
Before start
Library clean-ups should be done in a separate lab or area to the pre-PCR stage of the library preparation to prevent contamination.
Library clean up
Library clean up
16m 30s
16m 30s
Equilibrate Ampure XP beads to Room temperature for 00:30:00 and vortex well to mix.
Agencourt AmPure XP beadsCatalog #A63880
30m
If needed make libraries up to 50 µL with 10mM Tris pH8.
Add 45 µL AmpureXP to the samples (ratio 0.9:1) and mix well.
Note
We recommend a ratio of 0.9:1 Ampure:library to remove small unwanted fragments such as primer/adapter dimers from the library. Depending on the expected library size, the ratio can be increased for small libraries and decreased for large libraries. See Figure 1.
Figure 1. Size selection using different ratios of Ampure XP beads. Pink line shows the input sample with relative sizes of recovered DNA after incubation with 1.8X (red), 0.9X (blue), 0.7X (green) or 0.6X (cyan) volume of Ampure XP. Image taken from https://www.mybeckman.uk/reagents/genomic/cleanup-and-size-selection/pcr/performance.
Incubate at Room temperature room temperature for 00:05:00 .
Note
This time can be increased to improve recovery if yield is expected to be low.
5m
Place on a magnetic rack for 00:05:00 until beads and solution have fully separated.
Note
It is important to wait until the beads have fully separated, the time it takes will vary depending on the ratio of Ampure beads:sample, the level of DNA and the magnetic rack used.
5m
Carefully remove supernatant without disturbing the beads.
Note
The supernatant contains the small unwanted DNA fragments including primer/adapter dimers.
Carefully add 200 µL 80% Ethanol (freshly prepared) without disturbing the beads.
Incubate at Room temperature for 00:00:30 .
30s
Place on magnetic rack for 00:01:00 until the beads and solution have fully separated.
1m
Keeping on the magnet and carefully remove supernatant without disturbing the beads.
Repeat wash with 200 µL 80% Ethanol (freshly prepared) .
Remove all traces of Ethanol.
Note
Sample can be briefly centrifuged and placed back on the magnet to remove residual ethanol.
Keeping on the magnet air dry for up to 00:03:00 .
Note
It is important not to over dry the beads as this can interfere with DNA recovery, see Figure 2.
Figure 2. Examples of wet, dry and over dry beads. Image taken from https://www.ogt.com/resources/ngs-resources-support/ngs-tips-troubleshooting/ngs-success/purification-procedures/
3m
Add 18 µL 10mM Tris pH8 , remove from the magnet and mix well to fully suspend the beads.
Note
The elution volume can be adjusted as needed.
Note
If you plan on using the libraries for targeted enrichment with concentration by speedy vac then suspend the beads with nuclease-free water instead of 10 mM Tris pH8. For targeted enrichment with concentration by Ampure XP either nuclease-free water or 10 mM Tris pH8 can be used.
Incubate at Room temperature for at least 00:02:00 to elute the DNA.
2m
Place back on magnet until the beads and solution have fully separated.
Transfer the supernatant containing the library DNA to a fresh tube.
Library quantification
Library quantification
2m
2m
It is recommended that library quantification is performed using a fluorometric method (e.g. Qubit) rather than an absorbance based method (e.g. Nanodrop) as it is more specific, sensitive and accurate.
Equipment
Qubit
NAME
Flurometer
TYPE
Invitrogen
BRAND
Q33228
SKU
LINK
Prepare a 1:5 dilution of each sample by adding 2 µL library to 8 µL 10 mM Tris pH8 in fresh tubes.
Note
The dilution will depend on the expected yield of library. For accurate results the concentration in the diluted sample should vary between 1-50 ng/μL.
Prepare 0.5 mL thin-walled PCR tubes, including 2 tubes for the standard solution and 1 tube per sample. Label the tube lids and not the sides.
Prepare the Qubit dsDNA High Sensitivity master mix for the total number of samples and standards with an excess as follows:
| A | B | |
| Component | Volume (μl) | |
| Qubit dsDNA HS Buffer | 199 | |
| Qubit dsDNA HS Reagent | 1 | |
| Total | 200 |
Qubit® dsDNA HS Assay KitThermo Fisher ScientificCatalog #Q32854
Safety information
Qubit reagent is known to bind nucleic acid and is provided as a solution in DMSO. Treat the Qubit dsDNA HS Reagent with the same safety precautions as all other potential mutagens and dispose of the dye in accordance with local regulations.
Note
Ensure that the Qubit standards are equilibrated to room temperature for 30 min before use.
Aliquot the Qubit dsDNA High Sensitivity master mix into the assay tubes as follows:
Standards 190 µL Qubit master mix
Samples 199 µL Qubit master mix
Add 10 µL Qubit standard to each standard assay tube.
Add 1 µL diluted sample to each sample assay tube.
Vortex assay tubes and briefly centrifuge.
Incubate at Room temperature for 00:02:00 .
2m
Select dsDNA high Sensitivity assay on the Qubit Fluorimeter and press "Read Standards".
Insert Standard 1 and 2 into the sample chamber when prompted, close the lid and and press "Read Standard".
Note
It is recommended that you record the fluorescence values for the standards and monitor them to determine if there are any problems with your assay mix. The exact values will vary between machines and assay types.
Select "Run samples" and select the sample volume as 1 µL .
Insert a sample tube into the sample chamber, close the lid and press "Read tube".
Record the concentration of the Qubit sample in ng/μL.
Note
You may need to repeat the Qubit reading with a different sample dilution to be within range.
Calculate the concentration of the original sample by multiplying the Qubit concentration (see Step 31) by the dilution factor (see Step 19).
Library visualisation
Library visualisation
30m
30m
It is recommended that the libraries are visualised using capillary electrophoresis, we describe visualisation with a TapeStation and High Sensitivity D5000 ScreenTape. Alternatives such as the BioAnalyzer or Fragent Analyzer can also be used.
The purpose is to provide a size for the library fragments to give accurate molar quantification and to determine the quality of the library.
Equipment
4200 TapeStation System
NAME
Electrophoresis tool for DNA and RNA sample quality control.
TYPE
TapeStation Instruments
BRAND
G2991AA
SKU
LINK
Ensure that the D5000 ScreenTape and Reagents are equilibrated to Room temperature for at least 00:30:00 before use, vortex and briefly centrifuge.
High Sensitivity D5000 ScreenTapeAgilent TechnologiesCatalog #5067-5592
High Sensitivity D5000 ReagentsAgilent TechnologiesCatalog #5067-5593
Note
D5000 ScreenTape has a sizing range of 100-5000 bp. The D1000 ScreenTape and Reagents could be used as an alternative which has a sizing range of 35-1000 bp.
30m
Dilute each sample to approximately 1 ng/μL.
In fresh PCR strip tubes prepare the ladder assay tube as follows:
| A | B | |
| Component | Volume (μl) | |
| D5000 sample buffer | 2 | |
| D5000 ladder | 2 | |
| Total | 4 |
Prepare the sample assay tubes as follows:
| A | B | |
| Component | Volume (μl) | |
| D5000 sample buffer | 2 | |
| Diluted library | 2 | |
| Total | 4 |
Spin down, using IKA vortexer mix at 2000 rpm, 00:01:00 then spin down again.
1m
Load the assay tubes and ScreenTape into the TapeStation instrument.
Select the required sample/ladder positions in the TapeStation software and click "start".
Note
The TapeStation analysis software will open automatically at the end of the run to display the results.
Analyse the results a typical library should give a single peak of 200-500 bp depending on the library type.
Figure 3. Example profile for a typical Illumina library.
Note
See Troubleshooting section for examples of common problems.
Determine the library peak size in bp.
Note
If your library doesn't give a single sharp peak then a region size can be used instead.
Calculate the library molar concentration using the fragment size (Step 42) and sample mass concentration determined by Qubit (Step 32).
Library troubleshooting
Library troubleshooting
3m
3m
Below we list a number of common problems associated with Illumina sequencing libraries and steps you can take to rectify them.
Low yield
If your library yield is very low (e.g. less than 1 ng/μL) then the library can be amplified using the KAPA HiFi Library Amplification kit which uses primers directed against the Illumina adapter sequences.
KAPA HiFi HotStart Library Amplification Kit Illumina® PlatformsRocheCatalog #KK2620
Note
Be aware that excessive PCR cycles can lead to over amplification of reads and PCR duplicates.
Prepare the PCR mix (for multiple reactions a master mix with 10% excess is recommended):
| A | B | |
| Component | Volume (μl) | |
| 2X KAPA HiFi Ready Mix | 25 | |
| 10X Universal primer mix | 5 | |
| Total | 30 |
Add 30 µL PCR mix to fresh PCR tubes.
Add 20 µL sample .
Note
If necessary make sample up to 20 μL with 10 mM Tris pH8.
Incubate as follows on PCR machine:
98 °C for 00:00:45
1-6 cycles of
98 °C for 00:00:15
65 °C for 00:00:30
72 °C for 00:00:30
Final cycle of
72 °C for 00:01:00
4 °C hold .
3m
Repeat library clean up and QC (Steps 1-43).
go to step #1
Adapter dimers
Adapter dimers are small fragments (typically ~120-170 bp) that contain full length adapter sequences meaning that they are able to bind to the Illumina flow cell and be clustered. Due to the small size they cluster more efficiently than the full-length libraries meaning that it is very important that they are removed before sequencing.
Adapter dimers cause a number of problems:
- Reduce the number of library specific reads.
- Over clustering, reducing the data quality for all the samples.
- Shorter length than run length results in adapter dimer cluster giving no signal in later run cycles reducing quality.
- Free adapter can be incorporated into library clusters causing index hopping.
Figure 4. Example profile showing an Illumina sequencing library with prominant adapter dimer peak at 124 bp (A) and the same library after additional round of Ampure XP clean up (B).
Adapter dimers are removed by repeating the Ampure XP clean up (Steps 1-17)
go to step #1
If your specific library fragment size permits a more stringent ratio of Ampure:sample (e.g. 0.8X or 0.7X) can be used.
Depending on the extent of the adapter dimer, multiple rounds of clean up can be performed.
Note
An alternative to Ampure clean up is gel excision.
After you have completed your rounds of clean up repeat the QC (Steps 18-43)
go to step #18
Daisy chains
Assemblies of improperly annealed, partially double-stranded, hetero-duplex DNA called daisy chains can result from over amplification causing the depletion of primers/dNTP's in later cycles, or contaminants such as ethanol carried over from incomplete Ampure bead clean up into the final PCR reaction.
Figure 5. Example profile showing an Illumina sequencing library with larger daisy chain peaks (A) and the same library after additional PCR cycle (B).
Daisy chains can be broken up by performing 1-2 additional PCR cycles (Steps 45.1 - 45.5).
go to step #45.1
Large fragment contamination
Contamination with larger fragments are less serious than small fragment contamination and adapter dimers, as they cluster less efficiently than the smaller, specific libraries. They do interfere with accurate quantification and can be removed by reverse-ampure clean up.
Figure 6. Example profile showing an Illumina sequencing library showing larger fragment contamination.
Note
Reverse Ampure to remove larger fragments is less efficient than small fragment removal and will likely result in some loss of library.
If needed make libraries up to 50 µL with 10mM Tris pH8.
Add 30 µL AmpureXP to the samples (ratio 0.6:1).
Agencourt AmPure XP beadsCatalog #A63880
Note
Depending on the target library size this ratio can be reduced to 0.52:1 for more efficient large fragment removal.
Incubate at Room temperature room temperature for 00:05:00 .
Note
This time can be increased to ensure recovery if yield is expected to be low.
5m
Place on a magnetic rack for 00:05:00 until beads and solution have fully separated.
Note
It is important to wait until the beads have fully separated, the time it takes will vary depending on the ratio of Ampure beads:sample and the level of DNA.
5m
Carefully transfer the supernatant containing the smaller DNA fragments to fresh tubes.
Note
IMPORTANT: Unlike the previous clean ups it is the supernatant containing the smaller fragments NOT the beads containing the larger fragments that is retained.
Recover the DNA fragments form the supernatant by adding 60 µL AmpureXP (including the solution from step 48.2 this makes a ratio 1.8:1).
Incubate at Room temperature room temperature for 00:05:00 .
Note
This time can be increased to ensure recovery if yield is expected to be low.
5m
Continue with library clean up and QC as above (Steps 7-43).
go to step #7