TITLE: MAD4HatTeR  
AUTHORS: Andres Aranda-Diaz, eric.neubauervickers

[DESCRIPTION]
This protocol has been adapted from Paragon Genomics CleanPlex® NGS Panel

Information about the primer pools can be found here


[BEFORE_START]
A version of this protocol made an SOP in pdf format, including a didactic version, can be found at eppicenter.ucsf.edu/resources. That version may be more up to date.

[STEPS]
SECTION: mPCR
2. Add DNA 
SECTION: mPCR
3. Run PCR reaction on a thermocycler (in a separate room)
Initial denaturation: 95 °C 10 min 
Denaturation: 98 °C 15 s with ramping 3 °C 
Annealing/Extension: 60 °C 5 min with ramping 2 °C 
Repeat Denaturation and Annealing/Extension for X total cycles (see below). 
 
Hold at 10 °C 
SECTION: Reagent preparation
5. If you are not stopping at safe stopping point after first bead clean-up, also do this:


If using a 96-well pipettor, remove the tips corresponding to the empty wells (if any) on your plate. Save tips and put in used box for training purposes later.
SECTION: Reagent preparation
6. 
Proceed to next section within 30 min of finishing mPCR
SECTION: Post-mPCR bead purification
7. Spin the tubes/plate and add to each tube/well:
 2 µL 
 10 µL 

22 µL 
SECTION: Post-mPCR bead purification
10. Wash with 70% ethanol (use only freshly made 70% ethanol) 

SECTION: Post-mPCR bead purification
10.1. Add 180 µL 70 % volume ethanol 

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for 2 min or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells

SECTION: Post-mPCR bead purification
10.2. Remove the liquid with a P200 pipet

SECTION: Post-mPCR bead purification
10.5. Briefly spin down and remove the liquid leftovers using pipet set to 10-20 µL

PROTIP: Place the tubes in the spinner with the beads so that they are on the outside, further away from the center or axis of rotation. so that centrifuge force doesn't push them towards the opposite wall
SECTION: Post-mPCR bead purification
10.6. Leave tubes/wells open to dry Room temperature 
Generally, a 5 min dry time is enough, but it will depend on room temperature and humidity. The beads should look matte (right in figure), not shiny (left in figure). Under-drying (carrying ethanol) and over-drying (cracking) can lead to reduced yield

 

If you have only a few samples, you may want to keep an eye on each sample and close the tubes as they dry so that they all dry to the same extent.

SECTION: Post-mPCR bead purification
10.7. Add 10 µL 1X TE Close the tubes/wells and vortex vigorously to resuspend the beads. The magnetic beads will not affect the rest of the reactions, there is no need to remove them. 
Quickly spin down. 
SECTION: Post-mPCR bead purification
10.9. This is a safe stopping point
If you want to stop here, store -20 °C 

SECTION: Post-Digestion Purification
14. Perform a 1.3X bead purification  
Proceed immediately to indexing PCR reaction
SECTION: Indexing PCR reaction
16. Run PCR reaction on a thermocycler 
Initial denaturation: 95 °C 10 min 
Denaturation: 98 °C 15 s with ramping 3 °C 
Annealing/Extension: 60 °C 75 s with ramping 2 °C 
Repeat Denaturation and Annealing/Extension for 15 total cycles 
Hold at 10 °C 

SECTION: Pooling
24. Perform a 1X bead purification by adding magnetic beads to the combined sample.
Follow same steps as above but make sure that the volume of beads is the same than the pool volume
1X ratio should be 40 µL into 40 µL 

Elute into 40 µL 

SECTION: Capillary electrophoresis
25. Follow the instructions corresponding to the system you are using
SECTION: Gel purification
27. Cast a 2.5% agarose gel in TBE buffer with 1X SYBRsafe
SECTION: mPCR
1. Prepare mPCR mix and 
SECTION: mPCR
1.1. Thaw primer pools at Room temperature. Keep on ice after thawing

SECTION: mPCR
1.2. If expecting to run full protocol in one day, thaw CP Reagent Buffer (white tube)at Room temperature and keep on ice after thawing

SECTION: mPCR
1.3. Bring 5X mPCR Master Mix (green tube) into PCR Workstation in a cold rack
SECTION: mPCR
1.4. Combine the following volumes to prepare the mPCR reaction mix. Keep mix on ice or cold rack.
Vortex reagents to mix and briefly spin down before opening.

Generic recipe (without extra % for dead volumes):
 
     Reagent      Volume       …………..x No of reactions        5X   mPCR Master Mix (green tube)      2 µL               Each   primer pool      0.5   µL               Nuclease-free   H2O  If you are using less than 6 µL of   input DNA, increase water volume (e.g. if using 3 µL DNA, add water up to 7   µL)      Up to   4 µL          

SECTION: mPCR
1.5. Vortex mixes, briefly spin and keep on ice
SECTION: mPCR
1.6. Aliquot 4 µL (or 7 µL if using 3 µL input DNA) mPCR mix into PCR tubes/wells (single tubes, strips or plate).  Keep tubes on ice

SECTION: mPCR
1.7. Put primers and 5X mPCR Master Mix back in freezer

SECTION: mPCR
1.8. Transfer tubes outside of the PCR Workstation, to area where to add DNA 
SECTION: mPCR
2.1. Add 6 µL to each labeled tube/well, independent of parasitemia.
SECTION: mPCR
2.2. Close tubes or cover plate with seal. Vortex and spin down before proceeding 


10 µL
SECTION: Reagent preparation
4. Prepare for next steps
SECTION: Reagent preparation
4.1. Bring CleanMag Magnetic Beads and STOP buffer to Room temperature 
SECTION: Reagent preparation
4.2. Bring STOP buffer to room temperature and aliquot into PCR tube strip (~200 µL per tube) so that you can use a multichannel
SECTION: Reagent preparation
4.3. Make 70% ethanol with nuclease-free water (you will need 800 µL per sample)
SECTION: Reagent preparation
5.1. Bring index primers out of the freezer and thaw on ice 
SECTION: Reagent preparation
5.2. Make a plan for sample indexing. Write down what index you will use for each sample 
SECTION: Reagent preparation
5.3. Make Digestion Mix (If you are splitting the protocol in 2 days, make this mix at the beginning of the second day):
Perform this step in a PCR Workstation
on ice 
10 µL :
6 µL
2 µL 
2 µL 

* Buffer sometimes has a white precipitate after thawing. Make sure that it is completely dissolved before using

Make at least 10% extra for dead volume (e.g. for 10 samples, make 11 of the above)


SECTION: Reagent preparation
5.5. If using warm TE, move plate or tubes to  37 °C 
SECTION: Reagent preparation
5.4. Make Indexing PCR Mix (If you are splitting the protocol in 2 days, make this mix at the beginning of the second day):
Perform this step in a PCR Workstation

on ice
26 µL :
8 µL 
18 µL 

Make at least 10% extra for dead volume (e.g. for 10 samples, make 11 of the above)
SECTION: Post-mPCR bead purification
8. If you haven’t, prepare 70 % (v/v) ethanol with nuclease-free water
Make sure that CleanMag Magnetic Beads are at Room temperatureand are well mixed

SECTION: Post-mPCR bead purification
9.4. Remove all the liquid with a pipet set to >60 µL

SECTION: Post-mPCR bead purification
9.3. Briefly spin down and place on magnetic stand for 3 min or until the beads are collected on the side of the tubes/wells and the liquid is clear. 
SECTION: Post-mPCR bead purification
9.1. Add 1.3 times the volume of the mixture in CleanMag Magnetic Beads (29 µL for 1 primer pool, 31 µL for 2 pools)


SECTION: Post-mPCR bead purification
9. Incubate PCR products with beads

SECTION: Post-mPCR bead purification
9.2. Vortex vigorously to mix and incubate for 5 min at Room temperature

After this step, and until resuspension in TE, do not vortex and treat mixture carefully.
SECTION: Post-mPCR bead purification
9.5. Briefly spin down and remove the liquid leftovers using pipet set to 10-20 µL

PROTIP: Place the tubes in the spinner with the beads so that they are on the outside, further away from the center or axis of rotation. so that centrifuge force doesn't push them towards the opposite wall
SECTION: Post-mPCR bead purification
10.3. Repeat wash:
Add 180 µL 70 % volume ethanol 

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for 2 min or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells
SECTION: Post-mPCR bead purification
10.4. Remove the liquid with a P200 pipet
SECTION: Post-mPCR bead purification
10.8. Return TE to 37 °C 

SECTION: Digestion Reaction
11. If you stopped in the previous step and left samples at -20 °C   

SECTION: Digestion Reaction
12. Bring thermocycler block to 37 °C 

SECTION: Digestion Reaction
13. Perform Digestion reaction
SECTION: Digestion Reaction
13.1. Vortex Digestion reaction mix and briefly spin down to collect liquids
SECTION: Digestion Reaction
13.2. Add 10 µL to each tube/well.
SECTION: Digestion Reaction
13.3. Close tubes or seal plate (do not use a hot seal). Vortex and quickly spin down to collect liquids
SECTION: Digestion Reaction
13.4. Incubate at 37 °C for 10 min,. 
SECTION: Digestion Reaction
13.5. Immediately add 2 µL to each tube/well and mix by spinning briefly then vortexing. Spin again briefly to collect the liquid. 

22 µL 
SECTION: Indexing PCR reaction
15.2. Add to the 10 µL left in the tubes/wells:
26 µL 
2 µL and 2 µL . OR 4 µL (Make sure to centrifuge indexing primers before adding)
Indexing primers MUST only contribute to one well per primer

40 µL 


SECTION: Indexing PCR reaction
15. Mindexing PCR 
SECTION: Indexing PCR reaction
15.1. Vortex Indexing PCR mix and indexing primer plate and briefly spin down to collect liquids

SECTION: Indexing PCR reaction
15.3. Close tubes or seal plate
Vortex and briefly spin


SECTION: Capillary electrophoresis check
18. Briefly spin down tubes and place on magnetic stand to separate beads, which cannot be loaded into capillary electrophoresis systems
SECTION: Pooling
23. Briefly spin down tubes and place on magnetic stand to separate beads. 

Pool samples by mixing them into a single 1.5 mL microcentrifuge tube. 
We recommend skipping using the following volumes if using 15 cycles in mPCR:
30 µL
20 µL
15 µL
6 µL
3 µL


If you have capillary electrophoresis data for each of the samples, pool with volumes inversely proportional to the concentration of the 300-500 bp region
SECTION: Indexing PCR reaction
17. This is a safe stopping point
If you want to stop here, store -20 °C
SECTION: Capillary electrophoresis check
19. Select a random subset (8-16 depending on experiment design) of samples to run on capillary electrophoresis. Include negative and positive controls when available
SECTION: Capillary electrophoresis check
20. Follow the instructions corresponding to the system you are using
SECTION: Pooling
21. Before starting make 70% ethanol with nuclease-free water and bring CleanMag Magnetic Beads to room temperature
SECTION: Pooling
22. Create a sample sheet and double check that indexes are compatible
SECTION: Pooling
24.1. Add 1X the volume of the pool in CleanMag Magnetic Beads.

You may need to split in more than 1 tube if the total volume is > 1.5 mL
SECTION: Pooling
24.2. Vortex vigorously to mix and incubate for 5 min at Room temperature

After this step, and until resuspension in TE, do not vortex and treat mixture carefully.
SECTION: Pooling
24.3. Briefly spin down and place on magnetic stand for 3 min or until the beads are collected on the side of the tubes/wells and the liquid is clear. 
SECTION: Pooling
24.4. Remove all the liquid 
SECTION: Pooling
24.5. Briefly spin down and remove the liquid leftovers using pipet set to 10-20 µL

PROTIP: Place the tubes in the spinner with the beads so that they are on the outside, further away from the center or axis of rotation. so that centrifuge force doesn't push them towards the opposite wall
SECTION: Pooling
24.11. Briefly spin down and remove the liquid leftovers using pipet set to 10-20 µL

PROTIP: Place the tubes in the spinner with the beads so that they are on the outside, fur
SECTION: Pooling
24.6. Wash with 70% ethanol (use only freshly made 70% ethanol) 

SECTION: Pooling
24.7. Add 1 mL 70 % volume ethanol 

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for 2 min or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells
SECTION: Pooling
24.8. Remove all the liquid 
SECTION: Pooling
24.9. Repeat wash:
Add 1 mL 70 % volume ethanol 

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for 2 min or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells
SECTION: Pooling
24.10. Remove all the liquid 
SECTION: Pooling
24.12. Leave tubes/wells open to dry Room temperature 
Generally, a 5 min dry time is enough, but it will depend on room temperature and humidity.
SECTION: Pooling
24.13. Add 43 µL 1X TE taken from the incubator. Close the tubes/wells and vortex vigorously to resuspend the beads. 
If using multiple tubes, resuspend in one tube and use that resuspension to resuspend the rest of the tubes.
 
SECTION: Pooling
24.14. Quickly spin down and incubate at room Room temperature  for 2 min 

SECTION: Pooling
24.15. Place tube in magnetic stand and incubate at room temperature for 3 min or until liquid is clear

SECTION: Pooling
24.16. Transfer 40 µL to a clean, labeled tube

SECTION: Capillary electrophoresis
26. If there is no 150-250 bp peak or it is <5% proceed to loading the pool into sequencer
SECTION: Gel purification
28. Place the gel in an electrophoresis system and fill up with TBE buffer
SECTION: Gel purification
29. Load 5-10 µL DNA ladder to the first lane. Use a ladder that allows to distinguish 200 bp from 400 bp peaks.

SECTION: Gel purification
30. Add 8 µL 6X loading buffer to the 40 µL pool tube. Vortex and spin to collect liquids

SECTION: Gel purification
31. Load the pool into 1 or more lanes (depending on comb size you may not be able to fit in one lane). Leave an unused lane between the ladder and the pool
SECTION: Gel purification
32. Run with constant voltage at 140 V for 1 h
SECTION: Gel purification
33. After 1 h quickly image the gel. If there is a clear separation between primer dimers (~200 bp) and amplicons (~400 bp), you may continue to excise. Otherwise, run for longer
SECTION: Gel purification
34. Once the 2 bands are well separated, excise the 400 bp band
SECTION: Gel purification
35. Using a DNA gel extraction kit, dissolve the excised gel and run through a column following manufacturer’s instructions
SECTION: Gel purification
36. Elute with 15 µL elution buffer

SECTION: Capillary electrophoresis check
37. Follow the instructions corresponding to the system you are using
SECTION: Capillary electrophoresis check
38. If there is no 150-250 peak or it is <5% proceed to loading the pool into sequencer. Otherwise, you may need to run another gel extraction
SECTION: Sequencing
39. You are ready to sequence on an Illumina platform!