TITLE: DSBEST v2.0 (Caroe et al. 2018)
AUTHORS: Alicia Grealy

[DESCRIPTION]
This bench protocol is based on the work of Caroe et al. (2018), for preparing shotgun libraries from double-stranded DNA, typically for ancient and degraded DNA. Please cite Caroe et al. (2018) if you use this bench protocol!

[BEFORE_START]
I have begun including a double-stranded positive control oligo (dsCL104) in the library preparation; this is a double-stranded form of the single-stranded oligo described in Gansauge and Meyer (2013). Always include no-template controls and any extraction controls. 

Store PEG and ATP at -20 deg C and avoid repeated rounds of freeze thawing. 

Store MyOne C1 Streptavidin beads at 4 deg C in a fridge. 

[GUIDELINES]
Use at your own risk. You are responsible for double-checking that everything is correct!

Familiarise yourself with the relevent literature (Caroe et al. 2018) before attempting!

[STEPS]
SECTION: Preparation
1. 
 

"Suit up" in this order: hair net, nitrile gloves, facemask, coveralls, gumboots, booties, second pair of gloves.
SECTION: Preparation
2. Prepare the space by decontaminating surfaces with 10% household bleach followed by 70% ethanol. UV irradiate pipettes and racks. Racks should be bleached between subsequent uses and UV irradiated. 
SECTION: Preparation
3. Ensure ice is available. Thaw reagents on ice as needed. Keep enzymes on ice at all times. Do not vortex enzymes to mix but mix by flicking the tube gently. Pulse centrifuge all reagents before opening. 
SECTION: Preparation
4. Label tubes. 

 

SECTION: Preparation
5. Prepare all necessary buffers and UV decontaminate where appropriate. 
 

 

SECTION: Preparation
6. Before resuspending oligos, pulse centrifuge to collect the pellet at the bottom of the tube. Add the appropriate buffer (see Materials) and vortex thoroughly. Store at -20 deg C. Dilute out the working concentrations (below) and store at -20 deg C when not in use. Thaw on ice. Vortex and pulse centrifuge after each thaw. Before beginning library preparation, make sure you have enough of each working stock prepared!

 

 

SECTION: Preparation
7. Pre-program the thermal cycler.
SECTION: Prepare adapters
8. In a 0.2 ml Lo-bind PCR tube ("P5_DS1"), combine:
    
    40 ul of 500 uM IS1 
    40 ul of 500 uM IS3
    10 ul Ultrapure water
    10 ul 10X Oligo Hybridisation Buffer

Vortex and pulse centrifuge. 
SECTION: Prepare adapters
9. In a new 0.2 ml Lo-bind PCR tube ("P7_DS2"), combine:
    
    40 ul of 500 uM IS2
    40 ul of 500 uM IS3
    10 ul Ultrapure water
    10 ul 10X Oligo Hybridisation Buffer

Vortex and pulse centrifuge. 
SECTION: Prepare adapters
10. Incubate both "P5_DS1" and "P7_DS2" in a thermal cycler:
        
    95 deg C for 10 sec
    Ramp down to 12 deg C at a rate of 0.1 deg C/sec

SECTION: Prepare adapters
11. Combine both "P5_DS1" and "P7_DS2" together in a 0.5 ml Lo-bind Safelock tube to give 200 ul of 100 uM "DS_adapter_mix".
SECTION: Determine the DNA input amount and concentration of adapters required
12. Measure the concentration of your DNA extract on the Qubit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf


If possible, run the DNA on a fragment analyser or gel electrophoresis to determine the fragment length distribution.

https://www.perkinelmer.com/Content/LST_Software_Downloads/LabChip_GX_User_Manual.pdf
 







 

SECTION: Determine the DNA input amount and concentration of adapters required
13. Calculate the molarity of the dsDNA ends. Use the calculator at:

https://nebiocalculator.neb.com/#!/dsdnaends
 

SECTION: Determine the DNA input amount and concentration of adapters required
14. Calculate the pmol of adapters needed. This is approximately 10X the molarity of the dsDNA ends. Round up to the nearest 10 pmol.

This number is equal to the concentration of adapters needed in uM. 

Dilute the adapters to the desired working concentration in EBT buffer. 

 

 


SECTION: End Repair
15. Combine the following in a 1.5 ml Lo-bind Safelock tube. Vortex and pulse centrifuge. 


 

SECTION: End Repair
16. Aliquot 8 ul into a 0.2 ml Lo-bind PCR tube for each reaction. 
SECTION: End Repair
17. To make the total reaction volume up to 40 ul, add:

Up to 32 ul DNA to each sample reaction. Make up the remainder with Ultrapure water. Typically input 3x10^8 - 3x10^11 double-stranded molecules;  13 pg-14 ng of ca. 40 bp DNA--this is typically 20% of the extract. 

1 ul of 0.1 uM CL104_duplex positive control oligo to the positive conrol reaction + 31 ul Ultrapure water. We are inputing 3.01x10^10 molecules of single-stranded CL104 into the library preparation. 

32 ul of Ultrapure water to the no-template control reaction. 

Vortex and pulse centrifuge.
SECTION: End Repair
18. Incubate in a thermal cycler at:
        
       20 deg C for 30 min
       65 deg C for 30 min
       4 deg C hold
SECTION: Adapter ligation
19. Add 1 ul of the DS_adapter_mix DILUTION (e.g., 10 uM) to the finished end-repair reaction above. Vortex and pulse centrifuge. 
SECTION: Adapter ligation
20. Combine the following in a 0.5 ml Lo-bind tube. Vortex and pulse centrifuge. 
 

SECTION: Adapter ligation
21. Add 9 ul to each reaction. Vortex and pulse centrifuge. 
SECTION: Adapter ligation
22. Incubate in a thermal cycler at:

    20 deg C for 30 min
    65 deg C for 10 min
    4 deg C hold

SECTION: Fill in
23. Combine the following in a 0.5 ml Lo-bind tube. Vortex and pulse centrifuge. 
 

SECTION: Fill in
24. Add 10 ul of the above mix to each reaction. Vortex and pulse centrifuge. 
SECTION: Fill in
25. Incubate in a thermal cycler at:

    65 deg C for 15 min
    80 deg C for 15 min
    4 deg C hold
SECTION: Purify library
26. Purify the libraries using a QIAGEN MinElute PCR Purification kit.
 


https://www.qiagen.com/au/resources/resourcedetail?id=e24a0a3a-e9cb-4180-a6b4-202c527b924c&lang=en

Briefly...

SECTION: Purify library
27. Combine 300 ul of PB Buffer with each reaction in a 1.5 ml Lo-Bind tube. Vortex and pulse centrifuge. 
SECTION: Purify library
28. Transfer the mixture to a MinElute Silica Spin Column (purple) placed inside a collection tube. Centrifuge for 1 min at 13,000 rpm in a bench-top centrifuge. Discard the flow-through.
SECTION: Purify library
29. Add 700 ul of PE Buffer to the column. Centrifuge for 1 min at 13,000 rpm in a bench-top centrifuge. Discard the flow-through. 
SECTION: Purify library
30. Repeat Step 29.
SECTION: Purify library
31. Centrifuge one more time (dry) for 1 min at 13,000 rpm in a bench-top centrifuge. Place the column in a clean 1.5 ml Lo-bind tube with the lid cut off. 
SECTION: Purify library
32. Add 25 ul of EB buffer to the column. Incubate 5 min at room temperature.
SECTION: Purify library
33. Centrifuge 1 min at 13,000 rpm. Add the eluate back through the column to increase yield. Incubate for 5 min at room temperature. Centrifuge 1 min at 13,000 rpm.
SECTION: Purify library
34. Transfer the eluate to a clean 0.5 ml Lo-bind tube. 
SECTION: Purify library
35. Dilute the library 1 in 20 in Ultrapure water (i.e., add 1 ul of the library to 19 ul of Ultrapure water). Vortex and pulse centrifuge. 
SECTION: Purify library
36. Libraries can be stored at -20 deg C until amplification. For long-term storage, store at -80 deg C. 
SECTION: Quant the library
37. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 

 

SECTION: Quant the library
38. Make up the following master mix in a 1.5 ml Lo-bind tube. Vortex and pulse centrifuge. 

 

SECTION: Quant the library
39. Add 24 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes. 
SECTION: Quant the library
40. Add 1 ul of DNA sample to the corresponding PCR tubes according to the scheme below. Pulse centrifuge the tubes.


 

 

SECTION: Quant the library
41. Take the strip tubes to a post-PCR space. Place in thermal cycler and run the following program:

        95 deg C for 10 min

        Followed by 50 cycles of:
        
        95 deg C for 30 sec 
        60 deg C for 30 sec
        72 deg C for 30 sec
SECTION: Quant the library
42. Electrophorese 10 ul of the PCR product from the libraries (not standards) and controls on a 2% agarose gel. 

 

SECTION: Quant the library
43. Use the CT values from the qPCR to generate a standard curve for the standards in order to calculate how many template copies are present in each library. The positive control is used to calculate the efficiency of the library prep:

(# Copies of CL104 from Assay A / # copies CL104 from Assay B) * 100


 


This assay is also used to determine the number of cycles to give the indexing PCR, which needs to be stopped during the linear phase. 
SECTION: Index/amplify the library
44. Make up the following master mix in a 1.5 ml Lo-bind tube. Ensure to prepare enough master mix for 4 reactions per library plus pipetting error. Vortex and pulse centrifuge. 

 

 


 

SECTION: Index/amplify the library
45. Add 19 ul of master mix to the corresponding PCR tubes. Pulse centrifuge the tubes. 
SECTION: Index/amplify the library
46. Add  0.5 ul of the corresponding forward indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes. 
SECTION: Index/amplify the library
48. Add 5 ul of DNA sample to the corresponding reaction tubes according to the scheme below. Pulse centrifuge the tubes.

e.g., 
 


SECTION: Index/amplify the library
47. Add 0.5 ul of the corresponding reverse indexing primer to the appropriate reaction tube. Pulse centrifuge the tubes. 
SECTION: Index/amplify the library
49. Take the strip tubes to a post-PCR space. Place in qPCR machine and run the following program:

        95 deg C for 10 min
    
     Followed by _________ cycles of:
        
        95 deg C for 30 sec 
        60 deg C for 30 sec
        72 deg C for 30 sec


 

SECTION: Sequencing
62. Dilute the library to between 2-4 nM in Ultrapure water. 


SECTION: Purify the libraries
50. Pulse centrifuge the PCR tubes. Combine replicate PCR reactions into a 1.5 ml Lo-bine Safelock tube. Vortex and pulse centrifuge.
SECTION: Purify the libraries
51. Purify the libraries using SeraMag Speed Beads or SeraMag Select using a 1.6X beads : reaction volume (i.e., 160 ul). Follow the guidelines below:

https://www.gelifesciences.co.jp/catalog/pdf/SeraMagSelect_UserGuide.pdf

Elute in 35 ul of Ultrapure water. 
SECTION: Quantitate the libraries
52. Dilute the libraries 1 in 10 in Ultrapure water (i.e., 1 ul library in 9 ul Ultrapure water). 
SECTION: Quantitate the libraries
53. Use a LabChip GXII or equivalent fragment analyser (HiSense kit) to measure the molarity of the libraries between 160-500 bp. 

https://www.perkinelmer.com/Content/LST_Software_Downloads/LabChip_GX_User_Manual.pdf


 

SECTION: Pool libraries
54. Pool libraries in equimolar concentrations such that the total amount of DNA per library does not exceed 500-1000 ng. 
 

 

SECTION: Size select and purify
56. 
Run each pool in duplicate across two lanes (20 ul each) of a PippinHT electrophoresis system (2% gel, Marker 20B), selecting fragments between 160-500 bp and following the manufacturer's instructions:

http://www.sagescience.com/wp-content/uploads/2015/10/PippinHT-Operations-Manual-Rev-B_460005.pdf
SECTION: Pool libraries
55. Use a Vivaspin 500 (MWCO 30,000 Da) centrifugal column to concentrate each library to 20-40 ul. Centrifuge at 15,000 rcf with the membrane facing outwards for 30 sec at a time. 

https://www.sartorius.com/shop/ww/en/usd/applications-laboratory-filtration-ultrafiltration/vivaspin-500%2C-30%2C000-mwco-pes%2C-25pc/p/VS0121

Alternatively, concentrate the libraries using a SpeedyVac system, following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LED/manuals/DNA130%20User%20Manual%20final%20versionpdf.pdf
SECTION: Size select and purify
57. Combine replicates. Purify the libraries using SeraMag Speed Beads or SeraMag Select using a 2X beads : reaction volume (i.e., 160 ul). Follow the guidelines below:

https://www.gelifesciences.co.jp/catalog/pdf/SeraMagSelect_UserGuide.pdf

Elute in 25 ul of Ultrapure water. 
SECTION: Quantitate the final library
59. Measure the concentration of the neat library and these dilutions in duplicate on the Qubit following the manufacturer's instructions. 

https://assets.thermofisher.com/TFS-Assets/LSG/manuals/MAN0017209_Qubit_4_Fluorometer_UG.pdf
SECTION: Quantitate the final library
58. Dilute the libraries 1/2, 1/5, 1/10  in Ultrapure water (i.e., create a serial dilution in 10 ul volume).
SECTION: Quantitate the final library
60. Measure the molarity of the neat library and dilutions on a LabChip GXII Hisense kit (or equivalent fragment analyser) following the manufacturer's instructions:

https://www.perkinelmer.com/Content/LST_Software_Downloads/LabChip_GX_User_Manual.pdf
SECTION: Quantitate the final library
61. Based on the average fragment length and Qubit measurement, calculate the molarity of the library dilutions. Create a standard curve to check that the concentrations are linear. If they can be "trusted", extrapolate the neat concentration based on the dilutions. Average all the measurements of the neat concentration to get the best estimate of the library molarity.
SECTION: Sequencing
63. Follow the manufacturer's instructions to perform the sequencnig run on your platform of choice.