TITLE: Modified Illumina DNA Prep (M) Tagmentation Library Preparation for cDNA amplicons from wastewater
AUTHORS: Padmini   Ramachandran, Tamara  Walsky, Amanda  Windsor, Maria  Hoffmann, Chris   Grim

[DESCRIPTION]
This procedure outlines the protocol for sequencing of VarSkip Short SARS-CoV-2 cDNA amplicons using the Illumina DNA Prep library preparation kit for sequencing on an Illumina MiSeq sequencer. 



[BEFORE_START]
Run tape station or Bioanalyzer on the cDNA amplicon to determine the clean up of cDNA or no clean up as the next step. Quantify the cDNA using Qubit and determine the input cDNA concentration for the Illumina DNA prep.

[GUIDELINES]
 Illumina DNA Prep (M) Tagmentation Kit contain 3 components:
Box 1 of 3:
SPB (Store at 2-8oC)
TSB (Store at room temperature)
TWB (Store at room temperature)

Box 2 of 3: (Store at -25 to -15oC)
RSB
TB1
EPM

Box 3 of 3: (Store at 2-8oC)
BLT

Abbreviations:
BLT: Bead-Linked Transposome
dsDNA: Double-Stranded DNA
EPM: Enhanced PCR Mix
HT1: Hybridization Buffer
PCR: Polymerase Chain Reaction
PR2: Incorporation Buffer
RSB: Resuspension Buffer
SPB: Sample Purification Beads
TB1: Tagmentation Buffer 1
TSB: Tagment Stop Buffer
TWB: Tagment Wash Buffer

[STEPS]
SECTION: Dilute and Tagment Input DNA
2. Bring BLT (stored in refrigerator)  and TB1 (stored in freezer) to room temperature. 

Ensure that BLT is stored upright at all times, so that the beads remain submerged in the
buffer
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3. Label a 96-well PCR plate with the Run ID.

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4. Add molecular-grade water to the each sample well (from Column K of the workbook)

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5. Add gDNA to the molecular-grade water (per volume in Column J) and mix well by gently pipetting 5 - 10 times.
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6. Vortex BLT vigorously for 10 seconds, visually check the beads for complete resuspension and repeat vortexing if necessary. 

Do not spin down the BLT tube, the beads must be resuspended
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7. Vortex the TB1 and spin down the tube. 
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8. ***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***

Prepare the Tagmentation Master Mix: 

Combine 10 µL  of TB1 with 10 µL of BLT 

 
Note: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
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9. Vortex the tagmentation master thoroughly to make sure the BLT beads are evenly resuspended in the buffer.
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10. Using fresh tips, transfer 20 µL of tagmentation master mix to each sample well. 

Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet

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11. Pipette up and down 10 times mix the 50 μl reaction to resuspend the beads.
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12. Apply an adhesive PCR plate seal to the plate.
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13. Place the plate into the thermocyler and run the tagmentation program.

Program thermocyler to incubate  at 55 °C for 15 min  followed by a 10 °C hold with the lid heated at 100 °C
SECTION: Dilute and Tagment Input DNA
14. Check TSB for precipitate (if present, warm at 37 oC for up to 10 minutes and vortex) and ensure it is at room temperature prior to use.
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15. Upon completion of the incubation, remove the plate from the thermocycler. Proceed to the Post Tagmentation Cleanup step.
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16. Remove the plate seal.
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17. Add 10 µL of TSB to each sample ( A multi-channel pipette can be used) Gently pipette up and down 10 times to mix and fully resuspend the beads in the 50 μl reaction.

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18. Apply an adhesive PCR plate seal to the plate.
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19. Place the plate into the thermocyler and incubate at 37 °C for 15 min followed by a 10 °C hold with the lid heated at 100 °C 

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20. While samples are incubating, thaw EPM (stored in freezer) on ice and thaw indices at room temperature.
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21. Remove the plate from the thermocycler, quick spin the plate and remove the seal.
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22. Place the plate on a magnet for 3 min or until solution is clear.

Note: The DNA is tagged with adapters and bound to the beads.

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23. Using a multichannel pipette, remove the supernatant and discard.
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24. Remove the plate from the magnet and add 100 µLof TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended, try to avoid creation of foam from TWB.

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25. Place the plate on the magnet for 3 min or until solution is clear.

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26. Remove the supernatant and discard.
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27. Remove the plate from the magnet and add 100 µLof TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.

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28. Place the plate on the magnet for 3 min or until solution is clear.
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29. Remove the supernatant and discard.
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30. Remove the plate from the magnet and add 100 µLof TWB directly to the pellet. Gently pipette to mix until beads are fully resuspended.

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31. Place the plate with TWB on the magnet and allow to incubate until ready to proceed with adding the PCR master mix in the Amplify Tagmented DNA step. The plate should incubate for at least 3 minutes. It is important to keep the pellet in TWB to prevent overdrying of the beads.
SECTION: Amplification and Index Addition of Tagmented DNA
32. Invert the EPM to mix, then briefly centrifuge.
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33. Briefly centrifuge the Index plate.
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34. ***Scale up this step according to the number of reactions plus 3-4 for dead space volume/error***

Prepare the PCR master mix:

Combine 20 µL of EPM with 20 µL of Molecular grade water

  
Note: The 96 sample kit comes with 4 tubes of each reagent, each tube contains enough reagents for 24 samples
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35. Vortex and spin down the PCR master mix.
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36. Remove the third TWB wash from the samples while on the magnet. Remove any excess liquid from the plate using a small volume pipette.

Note: Removal of TWB is crucial, as it can impede PCR. Any foam remaining on the wells will not negatively impact the library.
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37. Remove the plate from the magnet and immediately proceed to adding the master mix.
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38. Add 40 µL of PCR master mix to each sample well. Gently pipette to mix to ensure beads are resuspended.

Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet

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39. Add 10 µL of the index primer pair from the appropriate index wells in accordance with the sample sheet. The plate has a foil seal on it, P20 tips are sufficient to pierce the seal to pipette. The indexes are single-use only.

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40. Use a pipette to gently mix a minimum of 10 times to ensure thorough mixing.
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41. Apply an adhesive PCR plate seal to the plate.
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43. Place the plate into the thermocycler and run the following pre-programmed settings with a heated lid at 100 °C 

    Thermocycler protocol (for use with DNA inputs above 100ng)
            Step 1: 68 °C  for 3 min 
            Step 2: 98 °C  for 3 min 
            Step 3: 5 cycles of:
                    98 °C  for 45 s 
                    62 °C  for 30 s 
                    68 °C  for 2 min 
            Step 4: 68 °C  for 1 min 
            Step 5: Hold at 10 °C 

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44. Centrifuge plate at 280 x g for 1 min 

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45. This is a safe stopping point. The plate may be sealed and stored at 2 °C to 
8 °C for up to 3 days.

SECTION: Clean up Libraries
46. NOTE: The steps listed below are critical for efficient size selection, product recovery and thus cluster generation and sequencing. Always check pipette tips for correct volumes and ensure that no beads have accidentally been aspirated. If beads have been aspirated or the bead pellet is disturbed, allow the pellet to reform (3-5 minutes on the magnet) and repeat the step.
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47. Before starting, prepare reagents: 
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47.1. Prepare fresh 80% ethanol sufficient for all samples. 
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47.2. Bring RSB to room temperature (from freezer) and vortex to mix.
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47.3. Bring SPB to room temperature (at least 30 minutes) from refrigerator. Vortex and invert SPB several times to full resuspend the beads.
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48.  Prepare the SPB Master mix:
  
Note: The ratio of SPB:water has been validated by FDA-CFSAN for size-selection.  CDC PulseNet uses a different ratio of SPB:water to select for large insert sizes. 
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49. If plate was retrieved from 4oC storage, centrifuge plate at 280 x g for 1 minute.

Remove the seal.
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50. Place the sample plate on the magnet for 5 min 

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51. Transfer 45 µLof supernatant (now containing the DNA) to a set of new wells on the sample plate.

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52. Remove sample plate from the magnet. 
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53. Vortex SPB master mix thoroughly and add 85 µL to each PCR product. 

Note: The master mix can be added to a reagent basin and distributed using a multichannel pipet
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54. Pipette to mix a minimum of 10 times or until thoroughly mixed.
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55. Incubate at room temperature for 5 min
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56. Place the 96-well plate on the magnet for 5 min or until supernatant is clear.

During incubation, vortex the stock SPB to resuspend the beads.

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57. With the plate still on the magnet, transfer 125 µL of supernatant (containing the DNA) to a new set of wells. 

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58. Remove the plate from the magnet and add 15 µL of stock SPB to the supernatant. 

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59. Gently pipet at least 10 times to mix. 
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60. Incubate at room temperature for 5 min

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61. Place on the magnet for 5 min or until clear.

Remove and discard the supernatant (DNA is now bound to the beads) without disrupting the beads
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62. Perform the steps below twice (for a total of two washes)
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62.1. While the plate is on the magnet, add 180 µL of the prepared 80% ethanol


Note: Do not add directly to the bead and do not mix.
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62.2. Incubate for 30 s

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62.3. Remove and discard ethanol.
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63. Use a pipette to remove any excess liquid from the plate.

Use a small volume pipette to get out any residual if necessary.
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64. Allow beads to air dry for up to  5 min (minimum of 3 minutes)

Note: Do not allow beads to over-dry. If bead pellet appears to be cracking, immediately resuspend beads regardless of drying time. 
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65. Remove the plate from the magnet and add 32 µL of RSB.  Pipet thoroughly to mix

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66. Incubate at room temperature for 2 min
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67. Place the plate on the magnet for 2 min
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68. Prepare a new 96-well plate as in below that is labelled with run date and intials. Transfer25 µL of the supernatant to a new 96-well plate. This is the final library.


   

SECTION: Clean up Libraries
69. If ready to proceed go to the Pooling Libraries step. Otherwise, this is a safe stopping point. The plate may be sealed and stored at -20 °C for up to 30 days.
SECTION: Quantification, Normalization and Pooling of Libraries
70. Quantify each sample using the Qubit dsDNA High Sensitivity kit. See SOP titled“DNA Quantification using the Qubit Fluorometer”  for more detailed information on performing DNA quantification.
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71. Enter the Qubit values into the "Prep Workbook" tab on the DNA Prep Library worksheet. The DNA Prep worksheet can be found in section 8 of this SOP. The dilution values will be calculated automatically.

SECTION: Quantification, Normalization and Pooling of Libraries
72. For v2 sequencing chemistry, it is recommended to dilute to 2nm; and for v3 sequencing chemistry to dilute to 4nm.
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73. Dilute each sample according to the values on the worksheet in the designated dilution wells on the 96-well plate.
SECTION: Quantification, Normalization and Pooling of Libraries
74. Pool 5 µL of each diluted library into the specified well and pipet to mix. Note: if sequencing Campylobacter, pool 2.5 µL of diluted sample.

SECTION: Denaturing Pooled LIbrary
75. Prepare a fresh aliquot of 0.2N NaOH. (This should be made fresh for each run)

Note: It is recommended to make aliquots of 1N NaOH and store in the freezer. 


SECTION: Denaturing Pooled LIbrary
76. Transfer 5 µL of the diluted library to a new Eppendorf LoBind tube.

SECTION: Denaturing Pooled LIbrary
77. Add 5 µL of 0.2N NaOH and pipette to mix.

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78. Incubate at room temperature for 5 min to denature the dsDNA. 

SECTION: Denaturing Pooled LIbrary
79. Immediately add 990 µLof HT1 and pipette to mix. The concentration for a 2 nm start is 10 pM and for 4 nm is 20 pM.

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80. Dilute the denatured library to the final desired loading concentration.  
  

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81. Mix by repeated inversion of sample tube. 

Optional: Denatured PhiX control can be spiked in at this point. 
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82. Heat the denatured DNA library to 96 °C  for 2 min in a heat block to ensure complete denaturation of all dsDNA in the sample. 

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83. Immediately cool in an ice-water bath for at least 5 min prior to loading. 

The DNA library may sit on ice or at 2 °C - 8 °C until ready for loading (<30 minutes)

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84. Proceed to loading 600 µL of the denatured ibrary to the thawed cartridge.

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85. Illumina MiSeq Operation and Maintenance: See “Procedure for Operation and Maintenance of the Illumina MiSeq for Whole Genome Sequencing” SOP for further instructions.
SECTION: Appendix
86. Appendix 1

DNA Prep Library Workbook
  



SECTION: Appendix
87. Appendix 2

Nextera DNA CD Indexes (96 indexes, plated)
  

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1. 
 

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42.