cDNA Synthesis

1. If beginning from isolated DNase treated viral RNA, cDNA synthesis can be performed using SSVILO or SSIV. This protocol will describe the SSIV protocol.

2. Combine the following components

3. Heat at 65°C for 5 min

4. Place samples on ice while making the SSIV Reaction Mix

5. Add the following to the annealed template RNA

6. Gently mix by pipetting and pulse spin the tubes to collect liquid at the bottom of the tube/plate.

7. Incubate the samples using the following parameters

8. Add 1µL of RNaseH to each sample

9. Incubate using the following parameters

10. Store samples at 4°C (for same day processing) or -20°C (up to a week) until ready for PCR.

Artic PCR - PCR generation of amplicons + Spike-in additions

11. Remove spike-in plate from the -20°C freezer and allow to thaw followed by a brief spin down.

12. Transfer 1 μL of 1 fMol spike-in into a new PCR strip/plate (if not using spike-in, just add 1µL additional water to ARTIC PCR Reaction)

13. Transfer 5 μL of cDNA into correspeonding well of PCR strip/plate

You will generate a 6 μL mix (5 μL cDNA + 1 μL spike-in)

NOTE: Viral Ct values can be used to normalize the 5 μL input to generate more uniform libraries.

An example of the Ct normalization scheme is described below:

All samples are normalized or diluted to a Ct of 27

1) The difference between 27 and the viral Ct value is first calculated and then rounded to the closest whole number

2) Calculations are based on the assumption of 100% PCR efficiency. As each PCR cycle results in a doubling of the input material, the doubling factor can be calculatated by taking the volume cDNA to be diluted (green box in B₂) and doubling this until you get to the desired cycle difference. The volume water to be added in C₂ should be the same number as the input cDNA volume in B₂.

The appropriate volume of water to be added for any cycle difference can then be calculated by adding up the number in the doubling factor column plus the total volume of water required from all previous cycles. For example a sample that needs to be diluted to a Ct of 27 from 24 would have a 3 cycle difference therefore 1 μL of sample would be added to 7 μL of water or if starting with 3 μL of sample, then 21 μL of water would be added.

Liquid handling devices can be used during this step to increase throughput

5 μL of the normalized cDNA can now be added to 1 μL of the spike in

14. Briefly mix and spin down - cDNA is now ready for ARTIC PCR with spike-in controls

15. Set up ARTIC PCR

16. Make master mix for Pool #1 and Pool #2 with all reagents except cDNA + Spike-in which have been prepared in a strip tube

17. Pipette 3 μL of cDNA + Spike-ininto a Strip tube/ plate labeled for pool #1. The remaining 3µL will stay in the original strip tube and be labeled pool #2

18. Add 22 μL of Pool #1 PCR mix to cDNA + Spike-in labeled pool #1

Mix by gentle vortex and quick spin down

19. Add 22 μL of Pool #2 PCR mix to cDNA + Spike-in labeled pool #2

Mix by gentle vortex and quick spin down

20. Run the Following PCR protocol ~ 4hr PCR program

21. NOTE: From this point on you are working with highly amplified DNA. Work should be done in an isolated area with extreme caution towards intra-sample contamination

Qubit ARTIC PCR

22. Prepare Qubit assay mix

23. Prepare Qubit standards: 10 μL of standard + 190 μL prepared reagent dye mastermix

24. Add 2 μL of sample + 198 μL of prepared reagent dye mastermix

Record qubit value for both pool #1 and pool #2 [NOTE: both pools should be roughly the same values]

Prepare Samples for Illumina DNA Flex [scaled down]

25. Ideal input range for scaled down FLEX is between 50ng -200ng of DNA

This should be an equal molar ratio of Pool #1 and Pool #2 from ARTIC PCR reaction

26. If Ct Normalization is done use the following table rather than normalizing again prior to Flex. Note: Just spot checking a couple samples is sufficient.

27. Total volume is 15 μL input into Flex

Prepare DNA Flex Reagent

28. Make sure to bring BLT and TB1 to room temp before use

Give a quick vortex and brief spin down

29. Add 10 μL of BLT/TB1 master mix using a multichannel into 15 μL for a total 25 μL reaction

Pipette up and down 10 times to mix

30. Incubate at Tagmentation Thermocycler conditions

31. Add 5 μL of TSB and pipette to mix. [Be careful when opening PCR strip tubes one at a time or with PCR plate removing seal]

Make sure the reaction is well mixed and beads are resuspended

32. Incubate under Tag STOP reaction

33. Remove from thermocycler and spin down plate briefly

WASH Beads

34. Place tubes/plate on magnet for 2 minutes or until clear

35. Remove and discard 30 μL of supernatant. Open the tubes one by one

(Tagmented DNA is attached to the beads) ***Be careful when discarding supernatant. It contains excess DNA.***

36. Remove tubes from magnet

37. Add 50 μL TWB to each well with a multi-channel pipette. Pipette to mix until fully resuspended

38. Place tubes on magnet for 2 minutes, or until clear

39. Remove and discard 50 μL of supernatant

40. Repeat wash steps 2 more times

41. Add 50 μL TWB to each well for 3^rd wash. Pipette to mix until fully resuspended.

42. Place tubes on magnet for 2 minutes, or until clear. Keep beads in TWB until ready to add PCR master mix.

Indexing PCR

43. Make PCR master mix with indexes

44. Remove Illumina index plate from -20°C freezer and defrost at room temperature. Give plate a quick spin before use.

45. Prepare indexes and PCR mix in seperate strip tube or plate

46. Add 20 μL of PCR mix to each well of a strip tube / plate

47. Add 5 μL of illumina index for each well

48. Index + PCR reaction is 25 μL

Gentle mix and briefly centrifuge down. Place on Ice til ready to use.

49. Remove 50 μL of wash supernatant from each tube

Briefly centrifuge and remove residual supernatant with a p20 pipette

50. Add 25 μL of PCR mix + index reaction to the tagmentation beads. Mix up and down gently

Give tubes/ plate a gentle spin down being carreful not to pellet the tagmentation beads

51. Run Indexing PCR

*SAFE STOPPING POINT - storage 4°C overnight or -20°C for longer term storage

Bead Clean up

Bring SPB beads to room temperature before use.

52. Place tubes on magnetic stand until clear

53. Transfer ~25 μL of supernatant to a new tube (DNA is in the supernatant)

54. Vortex SPB very well

55. Add 1.8X SPB to each sample based on volume [check several volumes of PCR reactions to confirm volumes for calculating 1.8x SPRI]

56. Pipette to mix 10 times

57. Incubate at room temperature for 5 minutes

58. Place the tubes on the magnet for 2 minutes

59. Remove and discard ~75 μL supernatant. (DNA is on the beads)

60. Keep tubes on magnet. Add 200 μL fresh 80% Ethanol to each sample

Time 30 seconds

61. Remove and discard 200 μL Ethanol from each sample

62. Repeat Ethanol wash 1x time

63. Removed residual ethanol wash with p20 pipette

64. Air dry beads with a gentle plastic cover for 5 minutes

65. Add 32 μL of RSB and pipette to resuspend

66. Incubate for 2 minutes

67. Place on magnet for 2 minutes or until clear

68. Transfer 30 μL into a new strip tube or plate

*SAFE STOPPING POINT - DNA library can be stored at 4°C for short term or -20°C for longer term storage

Quantification of library with Tape Station

69. Use 2 μL of sample and 2 μL of D1000 dye

70. Run on a D1000 tape and prepare final pooled sequeuncing library

Run library on a Miseq or Nextseq