TITLE: Library clean up and quality control for Illumina sequencing
AUTHORS: Katherine Smollett, Lily Tong, Kyriaki Nomikou, Jenna Nichols, Kirsty Kwok, Ma. Jowina Galarion, Daniel Mair, Ana Filipe

[DESCRIPTION]
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. 

[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.


[STEPS]
SECTION: Library clean up
1. Equilibrate Ampure XP beads to Room temperature for 30 min and vortex well to mix.
  

SECTION: Library clean up
2. If needed make libraries up to 50 µL  with 10mM Tris pH8.

SECTION: Library clean up
3. Add 45 µL to the samples (ratio 0.9:1) and mix well.

 


   

SECTION: Library clean up
4. Incubate at Room temperature room temperature for 5 min.

 


SECTION: Library clean up
5. Place on a magnetic rack for 5 min  until beads and solution have fully separated.

 

SECTION: Library clean up
6. Carefully remove supernatant without disturbing the beads.

 

SECTION: Library clean up
7. Carefully add 200 µL without disturbing the beads.
SECTION: Library clean up
12. Remove all traces of Ethanol.

 

SECTION: Library clean up
14. Add 18 µL, remove from the magnet and mix well to fully suspend the beads.

 

 


SECTION: Library clean up
9. Place on magnetic rack for 1 min  until the beads and solution have fully separated.

SECTION: Library clean up
11. Repeat wash with 200 µL.

SECTION: Library clean up
8. Incubate at Room temperature for  30 s.

SECTION: Library clean up
10. Keeping on the magnet and carefully remove supernatant without disturbing the beads.
SECTION: Library clean up
13. Keeping on the magnet air dry for up to 3 min.

 
  

SECTION: Library clean up
15. Incubate at Room temperature for at least  2 min to elute the DNA.

SECTION: Library clean up
16. Place back on magnet until the beads and solution have fully separated.
SECTION: Library clean up
17. Transfer the supernatant containing the library DNA to a fresh tube.


SECTION: Library quantification
18. 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.

 

SECTION: Library quantification
20. 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.

SECTION: Library quantification
21. Prepare the Qubit dsDNA High Sensitivity master mix for the total number of samples and standards with an excess as follows:

 

  
 


 


SECTION: Library quantification
22. Aliquot the Qubit dsDNA High Sensitivity master mix into the assay tubes as follows:

Standards 190 µL 
Samples 199 µL 

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

 


SECTION: Library quantification
23. Add 10 µL to each standard assay tube.

SECTION: Library quantification
24. Add 1 µL to each sample assay tube.

SECTION: Library quantification
25. Vortex assay tubes and briefly centrifuge.
SECTION: Library quantification
26. Incubate at Room temperature for 2 min.

SECTION: Library quantification
27. Select dsDNA high Sensitivity assay on the Qubit Fluorimeter and press "Read Standards".
SECTION: Library quantification
28. Insert Standard 1 and 2 into the sample chamber when prompted, close the lid and and press "Read Standard".

 

SECTION: Library quantification
29. Select "Run samples" and select the sample volume as 1 µL.

SECTION: Library quantification
30. Insert a sample tube into the sample chamber, close the lid and press "Read tube".
SECTION: Library quantification
31. Record the concentration of the Qubit sample in ng/μL.
 

SECTION: Library quantification
32. Calculate the concentration of the original sample by multiplying the Qubit concentration (see Step 31)  by the dilution factor  (see Step 19).

  

SECTION: Library visualisation
33. 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.

 

SECTION: Library visualisation
35. Dilute each sample to approximately 1 ng/μL.

SECTION: Library visualisation
34. Ensure that the D5000 ScreenTape and Reagents are equilibrated to Room temperature for at least 30 min before use, vortex and briefly centrifuge.
  
  


 

SECTION: Library visualisation
36. In fresh PCR strip tubes prepare the ladder assay tube as follows:


 

SECTION: Library visualisation
37. Prepare the sample assay tubes as follows:

 

SECTION: Library visualisation
38. Spin down, using IKA vortexer mix at 2000 rpm, 1 min then spin down again.

SECTION: Library visualisation
39. Load the assay tubes and ScreenTape into the TapeStation instrument.
SECTION: Library visualisation
40. Select the required sample/ladder positions in the TapeStation software and click "start".
 

SECTION: Library visualisation
41. Analyse the results a typical library should give a single peak of 200-500 bp depending on the library type. 

 
 


SECTION: Library visualisation
42. Determine the library peak size in bp.

 

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

  

SECTION: Library troubleshooting
44. Below we list a number of common problems associated with Illumina sequencing libraries and steps you can take to rectify them.
SECTION: Library troubleshooting
45. 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.
  

 

SECTION: Library troubleshooting
45.1. Prepare the PCR mix (for multiple reactions a master mix with 10% excess is recommended):

 


SECTION: Library troubleshooting
45.2. Add 30 µL to fresh PCR tubes.

SECTION: Library troubleshooting
45.3. Add 20 µL.

 


SECTION: Library troubleshooting
45.4. Incubate as follows on PCR machine:
98 °C for 45 s 
1-6 cycles of
98 °C for 15 s 
65 °C for 30 s 
72 °C for 30 s 
Final cycle of
 72 °C for 1 min 
4 °C .

SECTION: Library troubleshooting
45.5. Repeat library clean up and QC (Steps 1-43).

  

SECTION: Library troubleshooting
46. 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.

  
SECTION: Library troubleshooting
46.1. 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.

 

SECTION: Library troubleshooting
46.2. After you have completed your rounds of clean up repeat the QC (Steps 18-43)

  

SECTION: Library troubleshooting
47. 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.

  

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

  

SECTION: Library troubleshooting
48. 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.

  
 


SECTION: Library troubleshooting
48.1. If needed make libraries up to 50 µL  with 10mM  Tris pH8.
SECTION: Library troubleshooting
48.2. Add 30 µL to the samples (ratio 0.6:1).
  

 

SECTION: Library troubleshooting
48.3. Incubate at Room temperature room temperature for 5 min.

 

SECTION: Library troubleshooting
48.4. Place on a magnetic rack for 5 min  until beads and solution have fully separated.

 

SECTION: Library troubleshooting
48.5. Carefully transfer the supernatant containing the smaller DNA fragments to fresh tubes.
 

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

SECTION: Library troubleshooting
48.7. Incubate at Room temperature room temperature for 5 min.

 

SECTION: Library troubleshooting
48.8. Continue with library clean up and QC as above (Steps 7-43).