Library clean up and quality control for Illumina sequencing

Equilibrate Ampure XP beads to Room temperature for 0h 30m 0s and vortex well to mix.

If needed make libraries up to 50µL with 10mM Tris pH8.

Add 45µL to the samples (ratio 0.9:1) and mix well.

Incubate at Room temperature room temperature for 0h 5m 0s.

Place on a magnetic rack for 0h 5m 0s until beads and solution have fully separated.

Carefully remove supernatant without disturbing the beads.

Carefully add 200µL without disturbing the beads.

Incubate at Room temperature for 0h 0m 30s.

Place on magnetic rack for 0h 1m 0s until the beads and solution have fully separated.

Keeping on the magnet and carefully remove supernatant without disturbing the beads.

Repeat wash with 200µL.

Remove all traces of Ethanol.

Keeping on the magnet air dry for up to 0h 3m 0s.

Add 18µL, remove from the magnet and mix well to fully suspend the beads.

Incubate at Room temperature for at least 0h 2m 0s to elute the DNA.

Place back on magnet until the beads and solution have fully separated.

Transfer the supernatant containing the library DNA to a fresh tube.

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

Value	Label
Qubit	NAME
Flurometer	TYPE
Invitrogen	BRAND
Q33228	SKU

Prepare a 1:5 dilution of each sample by adding 2µL to 8µL in fresh tubes.

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

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 190µL

Samples 199µL

Add 10µL to each standard assay tube.

Add 1µL to each sample assay tube.

Vortex assay tubes and briefly centrifuge.

Incubate at Room temperature for 0h 2m 0s.

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".

Select "Run samples" and select the sample volume as 1µL.

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.

Calculate the concentration of the original sample by multiplying the Qubit concentration (see Step 31) by the dilution factor (see Step 19).

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

Value	Label
4200 TapeStation System	NAME
Electrophoresis tool for DNA and RNA sample quality control.	TYPE
TapeStation Instruments	BRAND
G2991AA	SKU

Ensure that the D5000 ScreenTape and Reagents are equilibrated to Room temperature for at least 0h 30m 0s before use, vortex and briefly centrifuge.

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.

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 2000rpm then spin down again.

Load the assay tubes and ScreenTape into the TapeStation instrument.

Select the required sample/ladder positions in the TapeStation software and click "start".

Analyse the results a typical library should give a single peak of 200-500 bp depending on the library type.

Determine the library peak size in bp.

Calculate the library molar concentration using the fragment size (Step 42) and sample mass concentration determined by Qubit (Step 32).

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.

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 30µL to fresh PCR tubes.

Add 20µL.

Incubate as follows on PCR machine:

98°C for 0h 0m 45s

1-6 cycles of

98°C for 0h 0m 15s

65°C for 0h 0m 30s

72°C for 0h 0m 30s

Final cycle of

72°C for 0h 1m 0s

4°C .

Repeat library clean up and QC (Steps 1-43).

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.

Adapter dimers are removed by repeating the Ampure XP clean up (Steps 1-17)

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.

After you have completed your rounds of clean up repeat the QC (Steps 18-43)

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.

Daisy chains can be broken up by performing 1-2 additional PCR cycles (Steps 45.1 - 45.5).

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.

If needed make libraries up to 50µL with 10mM Tris pH8.

Add 30µL to the samples (ratio 0.6:1).

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 0h 5m 0s.

Place on a magnetic rack for 0h 5m 0s until beads and solution have fully separated.

Carefully transfer the supernatant containing the smaller DNA fragments to fresh tubes.

Recover the DNA fragments form the supernatant by adding 60µL (including the solution from step 48.2 this makes a ratio 1.8:1).

Incubate at Room temperature room temperature for 0h 5m 0s.

Continue with library clean up and QC as above (Steps 7-43).