Jun 01, 2024

Public workspaceMAD4HatTeR V.4

DOI
dx.doi.org/10.17504/protocols.io.14egn779mv5d/v4
  • 1University of California, San Francisco;
  • 2Chan Zuckerberg Biohub
  • EPPIcenter
Andres Aranda-Diaz
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DOI: dx.doi.org/10.17504/protocols.io.14egn779mv5d/v4
Protocol Citation: Andres Aranda-Diaz, eric.neubauervickers 2024. MAD4HatTeR . protocols.io https://dx.doi.org/10.17504/protocols.io.14egn779mv5d/v4Version created by Andres Aranda-Diaz
Manuscript citation:
Aranda-Diaz A, Neubauer Vickers E 2022. MAD4HatTeR . protocols.io
https://dx.doi.org/10.17504/protocols.io.14egn779mv5d/v3
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: October 26, 2022
Last Modified: June 01, 2024
Protocol Integer ID: 101065
Abstract
This protocol has been adapted from Paragon Genomics CleanPlex® NGS Panel

Information about the primer pools can be found here

Safety warnings
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.
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.
mPCR
mPCR
Prepare mPCR mix and
Thaw primer pools at TemperatureRoom temperature . Keep TemperatureOn ice after thawing

If expecting to run full protocol in one day, thaw CP Reagent Buffer (white tube)at TemperatureRoom temperature and keep TemperatureOn ice after thawing

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

Generic recipe (without extra % for dead volumes):
ReagentVolume (µL)
5X mPCR Master Mix (green tube)2
Each primer pool0.5
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
Note: pools 1B and 2 are incompatible. Pools 3 and 4 are subsets of 1A and 5 is a subset of 1B. Incompatibilities also hold for subsets (5 and 2 are not compatible)
Pools are used at 0.25X for high parasitemia samples. When using more cycles (low parasitemia samples), halve primer concentration.

Vortex mixes, briefly spin and keep on ice
Aliquot Amount4 µL (or Amount7 µL if using 3 µL input DNA) mPCR mix into PCR tubes/wells (single tubes, strips or plate). Keep tubes TemperatureOn ice

Put primers and 5X mPCR Master Mix back in freezer

Transfer tubes outside of the PCR Workstation, to area where to add DNA
Add DNA
Add Amount6 µL DNA sample to each labeled tube/well, independent of parasitemia.
Close tubes or cover plate with seal. Vortex and spin down before proceeding


Amount10 µL final volume
Run PCR reaction on a thermocycler (in a separate room)
  • Initial denaturation: Temperature95 °C Duration00:10:00
  • Denaturation: Temperature98 °C Duration00:00:15 with ramping Temperature3 °C per second
  • Annealing/Extension: Temperature60 °C Duration00:05:00 with ramping Temperature2 °C per second
  • Repeat Denaturation and Annealing/Extension for X total cycles (see below).
Sample parasitemiaTotal number of cycles
≥100 p/µL15
<100 p/µL20
  • Hold at Temperature10 °C
Reagent preparation
Reagent preparation
Prepare for next steps
Bring CleanMag Magnetic Beads and STOP buffer to TemperatureRoom temperature
Bring STOP buffer to room temperature and aliquot into PCR tube strip (~200 µL per tube) so that you can use a multichannel
Make 70% ethanol with nuclease-free water (you will need 800 µL per sample)
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.
Bring index primers out of the freezer and thaw TemperatureOn ice
Make a plan for sample indexing. Write down what index you will use for each sample
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
TemperatureOn ice
Amount10 µL per reaction :
  • Amount6 µL H2O
  • Amount2 µL CP Reagent Buffer *
  • Amount2 µL CP Digestion Reagent

* 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)


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

TemperatureOn ice
Amount26 µL per reaction :
  • Amount8 µL 5X Second PCR Master Mix
  • Amount18 µL H2O

Make at least 10% extra for dead volume (e.g. for 10 samples, make 11 of the above)
If using warm TE, move plate or tubes to Temperature37 °C

Proceed to next section within Duration00:30:00 of finishing mPCR
Spin the tubes/plate and add to each tube/well:
  1. Amount2 µL STOP buffer (red tube)
  2. Amount10 µL 1X TE

Amount22 µL final volume
Step case

Mixing pools
70 steps

If you are mixing 2 mPCR reactions (e.g. mixing pools 1A/B and 2):
Spin the tubes/plate and add Amount4 µL STOP buffer to one of the tubes, change the volume in the pipet to 14 uL and transfer all of the volume to the other tube.

Amount24 µL final volume
If you haven’t, prepare Concentration70 % (v/v) ethanol with nuclease-free water
Make sure that CleanMag Magnetic Beads are at TemperatureRoom temperature and are well mixed

Incubate PCR products with beads

Add 1.3 times the volume of the mixture in CleanMag Magnetic Beads (Amount29 µL CleanMag Magnetic Beads for 1 primer pool, Amount31 µL CleanMag Magnetic Beads for 2 pools)


Vortex vigorously to mix and incubate for Duration00:05:00 at TemperatureRoom temperature

After this step, and until resuspension in TE, do not vortex and treat mixture carefully.
5m
Briefly spin down and place on magnetic stand for Duration00:03:00 or until the beads are collected on the side of the tubes/wells and the liquid is clear.
3m
Remove all the liquid with a pipet set to >60 µL

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
Wash with 70% ethanol (use only freshly made 70% ethanol)

Add Amount180 µL Concentration70 % volume ethanol ethanol

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for Duration00:02:00 or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells

2m
Remove the liquid with a P200 pipet

Repeat wash:
Add Amount180 µL Concentration70 % volume ethanol ethanol

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for Duration00:02:00 or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells
2m
Remove the liquid with a P200 pipet
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
Leave tubes/wells open to dry TemperatureRoom 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.

Add Amount10 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.
Return TE to Temperature37 °C

This is a safe stopping point
If you want to stop here, store Temperature-20 °C

Digestion Reaction
Digestion Reaction
If you stopped in the previous step and left samples at -20 °C Go to

Bring thermocycler block to Temperature37 °C

Perform Digestion reaction
Vortex Digestion reaction mix and briefly spin down to collect liquids
Add Amount10 µL Digestion Reaction Mix to each tube/well.
Close tubes or seal plate (do not use a hot seal). Vortex and quickly spin down to collect liquids
Incubate at Temperature37 °C for Duration00:10:00 ,.
10m
Immediately add Amount2 µL STOP Buffer to each tube/well and mix by spinning briefly then vortexing. Spin again briefly to collect the liquid.

Amount22 µL final volume
Post-Digestion Purification
Post-Digestion Purification
Perform a 1.3X bead purification Go to
Proceed immediately to indexing PCR reaction
Indexing PCR reaction
Indexing PCR reaction
Mindexing PCR
Vortex Indexing PCR mix and indexing primer plate and briefly spin down to collect liquids

Add to the Amount10 µL left in the tubes/wells:
  • Amount26 µL indexing PCR Mix
  • Amount2 µL forward indexing primer and Amount2 µL reverse indexing primer . OR Amount4 µL mixed indexing primers (Make sure to centrifuge indexing primers before adding)
  • Indexing primers MUST only contribute to one well per primer

Amount40 µL final volume


Close tubes or seal plate
Vortex and briefly spin


Run PCR reaction on a thermocycler
  • Initial denaturation: Temperature95 °C Duration00:10:00
  • Denaturation: Temperature98 °C Duration00:00:15 with ramping Temperature3 °C per second
  • Annealing/Extension: Temperature60 °C Duration00:01:15 with ramping Temperature2 °C per second
  • Repeat Denaturation and Annealing/Extension for 15 total cycles
  • Hold at Temperature10 °C

11m 30s
This is a safe stopping point
If you want to stop here, store Temperature-20 °C
Capillary electrophoresis check
Capillary electrophoresis check
Briefly spin down tubes and place on magnetic stand to separate beads, which cannot be loaded into capillary electrophoresis systems
Select a random subset (8-16 depending on experiment design) of samples to run on capillary electrophoresis. Include negative and positive controls when available
Follow the instructions corresponding to the system you are using
Pooling
Pooling
Before starting make 70% ethanol with nuclease-free water and bring CleanMag Magnetic Beads to room temperature
Create a sample sheet and double check that indexes are compatible
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:
  • Amount30 µL for 1 p/µL
  • Amount20 µL for 10 p/µL
  • Amount15 µL for 100 p/µL
  • Amount6 µL for 1,000 p/µL
  • Amount3 µL for 10,000 p/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
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 Amount40 µL CleanMag Magnetic Beads into Amount40 µL indexing PCR

Elute into Amount40 µL TE

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
Vortex vigorously to mix and incubate for Duration00:05:00 at TemperatureRoom temperature

After this step, and until resuspension in TE, do not vortex and treat mixture carefully.
5m
Briefly spin down and place on magnetic stand for Duration00:03:00 or until the beads are collected on the side of the tubes/wells and the liquid is clear.
3m
Remove all the liquid
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
Wash with 70% ethanol (use only freshly made 70% ethanol)

Add Amount1 mL Concentration70 % volume ethanol ethanol

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for Duration00:02:00 or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells
2m
Remove all the liquid
Repeat wash:
Add Amount1 mL Concentration70 % volume ethanol ethanol

To wash the beads, rotate the tubes/plate so that the beads migrate from one wall to the other. Incubate for Duration00:02:00 or until all beads have migrated. You may need to CAREFULLY flick the tubes/wells
2m
Remove all the liquid
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
Leave tubes/wells open to dry TemperatureRoom temperature
Generally, a 5 min dry time is enough, but it will depend on room temperature and humidity.
Add Amount43 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.
Quickly spin down and incubate at TemperatureRoom temperature for Duration00:02:00

2m
Place tube in magnetic stand and incubate at room temperature for Duration00:03:00 or until liquid is clear

3m
Transfer Amount40 µL to a clean, labeled tube

Capillary electrophoresis
Capillary electrophoresis
Follow the instructions corresponding to the system you are using
If there is no 150-250 bp peak or it is <5% proceed to loading the pool into sequencer
Gel purification
Gel purification
Cast a 2.5% agarose gel in TBE buffer with 1X SYBRsafe
Place the gel in an electrophoresis system and fill up with TBE buffer
Load Amount5-10 µL DNA ladder to the first lane. Use a ladder that allows to distinguish 200 bp from 400 bp peaks.

Add Amount8 µL 6X loading buffer to the Amount40 µL pool tube. Vortex and spin to collect liquids

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
Run with constant voltage at 140 V for 1 h
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
Once the 2 bands are well separated, excise the 400 bp band
Using a DNA gel extraction kit, dissolve the excised gel and run through a column following manufacturer’s instructions
Elute with Amount15 µL elution buffer

Capillary electrophoresis check
Capillary electrophoresis check
Follow the instructions corresponding to the system you are using
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
Sequencing
Sequencing
You are ready to sequence on an Illumina platform!