TITLE: ddRADSeq in a Field Setting
AUTHORS: Mrinalini Watsa, Gideon Erkenswick, Aaron Pomerantz, Stefan Prost

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This protocol begins with blood stored in Longmire's solution and has as its goal, the sequencing of genomic DNA from several individuals onto a single MinION sequencing run. It is based on a protocol outlined by Thrasher et al. (2018) for the same process on a large number of warbler samples, but it differs from this original protocol in a few key ways:</div><div class = "text-block"><ol style = "list-style-type: decimal;"><li style = "counter-reset:ol0;">Since we ultimately were targeting sequencing by a MinION sequencer, the ultimate processing steps reflect library preparation for runs on this device, instead of Illumina sequencers.</li><li style = "counter-reset:ol0;">For the same reason, we also size selected larger fragments than can be sequenced on a MiSeq platform.</li><li style = "counter-reset:ol0;">This protocol is also meant to only analysed a few individuals (20 or less) since the MinION read coverage is lower than that of a MiSeq.</li><li style = "counter-reset:ol0;">The entire protocol was carried out in a field laboratory in the southeastern Peruvian Amazon, and therefore many of the luxuries a institutional laboratory can afford were not present to us</li><li style = "counter-reset:ol0;">We used gel-based size selection in the absence of methods such as Blue Pippin.</li><li style = "counter-reset:ol0;">We stored blood on FTA cards and in Longmire's solution in our sampling program. We discovered that yields from FTA cards, regardless of including large numbers of hole punches per extraction, simply were too low for the sequencing goals of this project. Therefore, we include the protocol of a WGA amplification step for these low samples. Longmire's solution produced higher yields, but some times, multiple extractions might need to be pooled together with an SPRI bead cleanup to be fully effective. </li></ol></div></div>

[STEPS]
?. [P1 Adaptor Ligation]
Resuspend P1 oligos to 100uM in 0.5X AE Buffer (Qiagen), and follow instructions provided by supplier to do so if needed.
?. [P1 Adaptor Ligation]
In a PCR strip tube, mix equal volumes of each oligo together to get a final concentration of 50uM of adapter. E.g.  of P1F_X and l of P1R_X 11.
20 µl
20 µl
?. [P1 Adaptor Ligation]
In a  heat the mixture at  for
?. [P1 Adaptor Ligation]
Remove tube from the thermocylcer and place on a rack at .
[or room temperature]
?. [P1 Adaptor Ligation]
Allow to cool to  for at least .
[or room temperature]
?. [P1 Adaptor Ligation]
Further dilute the adapter to 5uM with 0.5X  in 1.5ml tube. E.g Add  of annealed adapter and  0.5X AE.
40 µl
360 µl
?. [P1 Adaptor Ligation]
Store adapters at or in freezer () for long term storage.
[minimum]
?. [P2 Adaptor Ligation]
5In a PCR tube mix equal volumes of each oligo together to get a final concentration of 50uM of adapter. E.g.  of P2F_MspI and  of P2R_MspI.
50 µl
50 µl
?. [P2 Adaptor Ligation]
In a thermocylcer heat the mixture at  for
?. [P2 Adaptor Ligation]
Remove tube from the thermocylcer and place on a rack at .
[or room temperature]
?. [P2 Adaptor Ligation]
Allow to cool to  for at least .
[or room temperature]
?. [P2 Adaptor Ligation]
Further dilute the adapter to 25uM with 0.5 X  in 1.5ml tube. E.g Add  of annealed adapter and  0.5X AE.
100 µl
100 µl
?. [P2 Adaptor Ligation]
Store adapter at  or in freezer for long term storage.
?. [P1 1:20 Dilution]
This can be done in a strip of PCR tubes to allow you to use the multichannel pipette to dispense the P1 adapters. (You will need 20 tubes if using all the P1 adapters)
?. [P1 1:20 Dilution]
Label tubes.
?. [P1 1:20 Dilution]
Add  of nuclease free water to each tube.
19 µl
?. [P1 1:20 Dilution]
Add  of P1 adapter to corresponding one tube above.
1 µl
?. [P1 1:20 Dilution]
Repeat for all P1 adapters being used.
?. [P1 1:20 Dilution]
Seal tubes, vortex briefly to mix, centrifuge.
?. [FTA Card Extractions]
Add  of ddH2O to each tube.
500 µl
?. [FTA Card Extractions]
Rinse each sample in ddH2O by vortexing three times for  each
?. [FTA Card Extractions]
Using a sterile tip, transfer each disk to a pre-labeled 0.8 mL PCR tube.
?. [FTA Card Extractions]
Add  to of ddH2O so that it completely covers the disk
45 µl
50 µl
?. [FTA Card Extractions]
Spin down tubes and make sure disk(s) are submerged
?. [FTA Card Extractions]
Heat to  for  in a PCR machine
?. [FTA Card Extractions]
Remove tube and pulse vortex or gently tap 60 times.
?. [FTA Card Extractions]
Centrifuge for
?. [FTA Card Extractions]
Transfer all liquid into a pre-labelled tube – this will be Elution A
?. [FTA Card Extractions]
Add  of ddH20 to the disk that remains in the PCR tube and repeat steps 5 - 10 to get Elution B
30 µl
?. [FTA Card Extractions]
Take 1 to 3 (2-3 mm3) punches from an FTA card using a fine scalpel or a hole punch, while maintaining sterility.  Transfer it into a 1.5 mL microcentrifuge tube.
?. [FTA Card DNA Extractions]
2.	Rinse each sample in ddH2O by vortexing three times for five seconds each
?. [FTA Card DNA Extractions]
3.	Using a steril tip, transfer each disk to a pre-labeled PCR tube.
?. [FTA Card DNA Extractions]
4.	Add 45 to 50 uL of ddH2O (should cover the disk)
?. [FTA Card DNA Extractions]
5.	Spin down tubes and make sure disk is submerged
?. [FTA Card DNA Extractions]
6.	Heat to 95°C for 30 minutes in a PCR machine (
?. [FTA Card DNA Extractions]
7.	Remove tube and pulse vortex or gently tap 60 times.
?. [FTA Card DNA Extractions]
8.	Centrifuge for 30 seconds
?. [FTA Card DNA Extractions]
9.	Transfer all liquid into a pre-labelled tube – this will be Elution A
?. [FTA Card DNA Extractions]
10.	Add 45 to 50 uL (Confirm that you did use 30 ul for each elution) to the disk that remains in the PCR tube and repeat steps 4 – 9 to get Elution B
?. [FTA Card DNA Extractions]
11.	Store at 4°C if using it soon, or store at -20°C otherwise
?. [FTA Card Extractions]
Store at  if using it soon, or store at  for longterm storage.
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 1]
Vortex sample for
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 1]
Add  of Proteinase K
10 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 1]
Incubate overnightat  -. The digest iscomplete when the colour of the supernatant has turned light orange
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Add  of Binding Buffer to the tube containing 200 uL of Longmire's solution with blood.
500 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Pulse vortex for
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Incubate for  at -
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Add  of 96% ethanol and pulse vortex 5 times
500 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Centrifuge at 16000 G for
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Carefully transfer as much supernatant as possible to the spin column, loading  at a time, centrifuge at 6000 G for  and discard the liquid.
500 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Centrifuge at 6000 G for  and discard the liquid.
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Add  of Wash Buffer, centrifuge at 6000 G for  and discard the liquid.
720 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Centrifuge again at 10,000 G for  and discard the liquid.
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Remove spin column and place into a labelled microcentrifuge tube.
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Incubate tubes with caps off at  to evaporate residual ethanol for .
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
During this incubation, place  of elution buffer into the bath to heat it.
[or sufficient amount]
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Add  of elution buffer directly onto the membrane of each spin column.
20 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Incubate at roomtemperature for .
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Repeat steps 28 to 30 with  of elution buffer for a second elution B. If you like, you can also elute this directly into Elution A for one tube at the end.
30 µl
?. [DNA Extractions from Blood Stored in Longmire Solution: Day 2]
Centrifuge at 10,000 G for to collect eluted DNA.
?. [Quantify DNA]
Choose the OneDNA protocol on the Quantus fluorometer.
?. [Quantify DNA]
Calibrate the Quantus as follows: a.	Fill a Qubit assay tube with  dye and click ‘Blank’ (under ‘Calibrate).
200 µl
?. [Quantify DNA]
Prepare a labeled Qubit assay tube for each sample to be quantified
?. [Quantify DNA]
Add  of dye to each Qubit assay tube.
200 µl
Critical Note: tubes containing dye need to be covered with aluminium foil to shield dye from light.
?. [Quantify DNA]
Vortex the samples to mix them well and quickly spin down.
?. [Quantify DNA]
Add of each sample to each labeled Qubit assay tube.
1 µl
?. [Quantify DNA]
Vortex and spin assay tubes, and incubate at  for .
[or rtp]
?. [Quantify DNA]
Measure the sample in the fluorometer and record value in ng/uL.
We have reused Quantus assay tubes after a recycling program. Rinse each tube with 10% bleach, two washes in distilled water, and 70% ethanol. Then autoclave. These tubes should be measured once without the sample, for some of them will have a very small amount of residual DNA. Then, add the sample, incubate for 5 minutes, and re-measure. Subtract the original reading from this one for an accurate reading. This method allows us to recylce the plastic tubes without detriment to our accuracy.
?. [Quantify DNA]
b.	In a new Qubit assay tube, add of dye and  of Lambda DNA, and click ‘Standard’. Then click 'Save'
400 µl
2 µl
?. [Whole Genome Amplification ]
Prepare and label one PCR tube for each sample to be amplified using the (NAME KIT)
?. [Whole Genome Amplification ]
Add of sample buffer (Green cap) to each tube.
9 µl
?. [Whole Genome Amplification ]
Dilute template DNA to 10 ng/uL. Add  of this DNA to the PCR tube.
1 µl
If DNA concentration doesn't reach 10ng/uL, then add more. We added 3 µl of our template to each tube because our DNA concentration was too low.
?. [Whole Genome Amplification ]
Pulse vortex the PCR tubes.
?. [Whole Genome Amplification ]
Heat the samples at  for  and then cool to .
Heating DNA for longer than 3 minutes at higher temperatures can cause damage to the DNA.
?. [Whole Genome Amplification ]
While waiting for samples to cool, prepare a Master Mix: For each amplification reaction, combine of Reaction Buffer (Blue cap) with of Enzyme Buffer (Yellow cap).
9 µl
1 µl
Prepare the Master Mix right before you use it and keep in on ice. If there is any left over after WGA, do not reuse it.
Our enzyme:buffer concentration was 1:5.6, rather than 1:9 (by accident). This did not appear to have an effect on the amplification.
?. [Whole Genome Amplification ]
Transfer  of the Master Mix to each sample.
10 µl
?. [Whole Genome Amplification ]
Incubate at  for . If you had low concentration to start with, this can be extended for 30 more minutes.
?. [Whole Genome Amplification ]
Inactivate samples at  for .
?. [Whole Genome Amplification ]
Pause point: Samples can be stored at 4° C.
?. [Primer 1 Adaptor Ligation]
Resuspend oligos to 100uM in 0.5X AE Buffer (Qiagen), using supplier's instructions if needed.
?. [Primer 1 Adaptor Ligation]
In a PCR tube mix equal volumes of each oligo together to get a final concentration of 50uM of adapter. E.g. 20ul of P1F_X and 20ul of P1R_X 11.
?. [Primer 1 Adaptor Ligation]
In a thermocylcer heat the mixture at  for
?. [Primer 1 Adaptor Ligation]
Remove tube from the thermocylcer and place on a rack at .
[or rtp]
?. [Primer 1 Adaptor Ligation]
Allow to cool to  for at least .
[or rtp]
?. [Primer 1 Adaptor Ligation]
Further dilute the adapter to 5uM with 0.5 X AE in 1.5ml tube. E.g Add  of annealed adapter and  0.5X AE.
40 µl
360 µl
?. [Primer 1 Adaptor Ligation]
Store adapters at  or in freezer for long term storage.
?. [Primer 1 Dilution]
Label tubes. These dilutions can be done in a strip of PCR tubes to allow you to use the multichannel pipette to dispense the P1 adapters. (You will need 20 tubes if using all the P1 adapters)
?. [Primer 1 Dilution]
Add  of nuclease free water to each tube.
19 µl
?. [Primer 1 Dilution]
1Add  of P1 adapter to corresponding one tube above.
1 µl
?. [Primer 1 Dilution]
Repeat for all P1 adapters being used.
?. [Primer 1 Dilution]
Seal tubes, vortex briefly to mix, centrifuge.
?. [Primer 2 Adaptor Ligation]
In a PCR tube mix equal volumes of each oligo together to get a final concentration of 50uM of adapter. E.g. 50ul of P2F_MspI and 50ul of P2R_MspI.
?. [Primer 2 Adaptor Ligation]
In a thermocylcer heat the mixture at  for
?. [Primer 2 Adaptor Ligation]
Remove tube from the thermocylcer and place on a rack at
[or rtp]
?. [Primer 2 Adaptor Ligation]
Allow to cool to  for at least .
[or rtp]
?. [Primer 2 Adaptor Ligation]
Further dilute the adapter to 25uM with 0.5 X AE in 1.5ml tube. E.g Add 100ul of annealed adapter and 100ul 0.5X AE.
?. [Primer 2 Adaptor Ligation]
Store adapter at or in a freezer for long term storage.
?. [Barcoding and Pooling Strategy]
Choosing how to pool your samples is dependent on the number of samples involved and how successful you have been in each case in extracting DNA from the samples. Additionally, the number of dual indices you create will inform the maximum number of samples that can be pooled together. As such, there are a few general guidelines to follow:1. Make sure you combine samples in the same pool that have roughly the same DNA output. i.e. pool all samples with the same concentration (as per your fluorometer reading) together. 2. When choosing indices, follow the barcoding system utilised in Thrasher et al. 2018 if you plan to do Illumina-platform sequencing (i.e. the MiSeq or HiSeq). If you are utilising a MinION or other 3rd gen sequencer with a higher error rate, demultiplexing your samples becomes very difficult if you use barcodes as short as the ones in Thrasher et al. 2018. We strongly encourage you to extend the length of barcodes to more easily distinguish samples from each other bioinformatically post sequencing.
?. [Barcoding and Pooling Strategy]
Once you have pooled your samples together,  you will need 20uL of your pools, containing ~ 400 ng of DNA each. If you do not have this concentration, but you have this amount of DNA in total, consider doing an SPRI bead cleanup to concentrate your DNA so you are left with this amount in the end. If you simply do not have enough DNA, consider a second extraction combined with this one via a SPRI bead cleanup to reach these targets. Without this much DNA, you might have a lower yield of reads, although it is not impossible to achieve desired results. We recommend a bare minimum of 100 ng of DNA for any single sample.
?. [Double Restriction Enzyme Digest]
Make a master mix of everything except the P1 adapters and DNA in a 2ml tube. Make the master mix for a few extra reactions so that you do not run out. Use the ligation mix table below. If you do not have those exact concentrations, then use the table from the next step instead:  AB1ReagentsVolume  (uL) for one reaction2Nuclease free water6.25310X CutSmart Buffer4425uM P2 adaptor2.5510mM ATP4620U/uL Sbfl-HF0.75720U/uL MspI0.758400U/uL T4DNA ligase0.759Total19
AB1ReagentsVolume  (uL) for one reaction2Nuclease free water6.25310X CutSmart Buffer4425uM P2 adaptor2.5510mM ATP4620U/uL Sbfl-HF0.75720U/uL MspI0.758400U/uL T4DNA ligase0.759Total19
?. [Double Restriction Enzyme Digest]
If you do not have those exact concentrations, then use the table below:  AB1ReagentsVolume  (uL) for one reaction2Nuclease free waterUp to total volume310X CutSmart BufferDilute to 1X425uM P2 adaptor2.5510mM ATPDilute to 1mM620U/uL Sbfl-HF0.75720U/uL MspI0.758400U/uL T4DNA ligase0.759Normalized DNAApprox. 200-500ng10Total19
AB1ReagentsVolume  (uL) for one reaction2Nuclease free waterUp to total volume310X CutSmart BufferDilute to 1X425uM P2 adaptor2.5510mM ATPDilute to 1mM620U/uL Sbfl-HF0.75720U/uL MspI0.758400U/uL T4DNA ligase0.759Normalized DNAApprox. 200-500ng10Total19
?. [Double Restriction Enzyme Digest]
Vortex briefly to mix and centrifuge.
?. [Double Restriction Enzyme Digest]
Aliquot 19 ul of master mix to each well of a PCR strip tube or plate (depending on the size of your reaction)
?. [Double Restriction Enzyme Digest]
Add 1ul of P1 adapter to the corresponding well of the PCR plate (make sure that each pair of 2 digestion-ligation reactions receives a unique P1 adapter).
?. [Double Restriction Enzyme Digest]
Add 20ul of gDNA to each well.Note: every well will have a different DNA sample.
?. [Double Restriction Enzyme Digest]
Use a pipette set to 30ul and gently pipette up and down a few times to mix each reaction.
?. [Double Restriction Enzyme Digest]
Seal each PCR strip tube well, making sure the snap caps are down and that the contents of the tube are settled at the bottom.
?. [Double Restriction Enzyme Digest]
Incubate at the following temperatures and times:  for  for  hold
?. [Gel-based Size Selection and Purification]
1. Set a thick 1% agarose gel. For the MiniOne PCR system, we used ~19 mL agarose / gel + 1.5 uL dye, and the 6-toothed comb. Our gels could hold up to 35uL of product
?. [Gel-based Size Selection and Purification]
Load your samples into wells along with a 100bp ladder. Samples may have to be loaded into multiple wells.
Critical Note: Multiple samples can be run on the same gel, but to prevent contamination across lanes, only run samples that will be pooled into the same pool on the same gel.We additionally reused 1x TB buffer for gels containing samples from a common pool.
?. [Gel-based Size Selection and Purification]
Run until the desired size can be clearly distinguished from the ladder.
We ran gels for ~25 minutes until the 1000 bp band could be clearly distinguished from the 1500 bp band on the ladder.
?. [Gel-based Size Selection and Purification]
Take and label photo of gel for future reference.
?. [Gel-based Size Selection and Purification]
The following steps are based on the gel-extraction kit you use. We used this kit
?. [Gel-based Size Selection and Purification]
Prepare an empty 1.5 mL microcentrifuge tube and record its weight.
?. [Gel-based Size Selection and Purification]
Using one gel excision tip, cut out the region surrounding desired size as cleanly as possible for each lane and combine all gel slices in the microcentrifuge tube.
Take and label photo of excised gel for future reference.For all samples, we excised the region surrounding the 500 bp and the 1000 bp band.
?. [Gel-based Size Selection and Purification]
Re-weigh microcentrifuge tube and record.
?. [Gel-based Size Selection and Purification]
[Repeat for all samples in your dataset]
?. [Gel-based Size Selection and Purification]
Add Membrane Binding Solution at a ratio of 10μl of solution per 10mg of agarose gel slice. Vortex the mixture
?. [Gel-based Size Selection and Purification]
Incubate at for  or until the gel slice is completely dissolved.
Note: Vortex the tube every few minutes to increase the rate of agarose gel melting.
?. [Gel-based Size Selection and Purification]
Centrifuge the tube briefly at room temperature to ensure the contents are at the bottom of the tube.
Note: Once the agarose gel is melted, the gel will not resolidify at room temperature.
?. [Gel-based Size Selection and Purification]
Place one SV Minicolumn in a Collection Tube for each dissolved gel slice.
?. [Gel-based Size Selection and Purification]
Transfer the dissolved gel mixture to the SV Minicolumn assembly and incubate for  at room temperature.
?. [Gel-based Size Selection and Purification]
Centrifuge the SV Minicolumn assembly in a microcentrifuge at 16,000 × g (14,000 rpm) for .
?. [Gel-based Size Selection and Purification]
Remove the SV Minicolumn from the Spin Column assembly, and discard the liquid in the Collection Tube.
?. [Gel-based Size Selection and Purification]
Return the SV Minicolumn to the Collection Tube.
?. [Gel-based Size Selection and Purification]
Wash the column by adding of Membrane Wash Solution to the SV Minicolumn.
700 µl
?. [Gel-based Size Selection and Purification]
Centrifuge the SV Minicolumn assembly for  at 16,000 × g (14,000rpm).
?. [Gel-based Size Selection and Purification]
Empty the Collection Tube as before, and place the SV Minicolumn back in the Collection Tube.
?. [Gel-based Size Selection and Purification]
Repeat the wash with  of Membrane Wash Solution, and centrifuge the SV Minicolumn assembly for  at 16,000 × g.
500 µl
?. [Gel-based Size Selection and Purification]
Remove the SV Minicolumn assembly from the centrifuge, being careful not to wet the bottom of the column with the flowthrough.
?. [Gel-based Size Selection and Purification]
Empty the Collection Tube, and recentrifuge the column assembly for  with the microcentrifuge lid open (or off) to allow evaporation of any residual ethanol.
?. [Gel-based Size Selection and Purification]
Carefully transfer the SV Minicolumn to a clean 1.5ml microcentrifuge tube.
?. [Gel-based Size Selection and Purification]
Apply  of nuclease-free water directly to the center of the column without touching the membrane with the pipette tip.
20 µl
Alternatives: You can use an elution buffer of your choice here instea of nuclease-free water, depending on your downstream applications
?. [Gel-based Size Selection and Purification]
Centrifuge for  at 16,000 × g (14,000 rpm).
?. [Gel-based Size Selection and Purification]
Remove the eluted volume and place it back on the spin column.
?. [Gel-based Size Selection and Purification]
Centrifuge for  at 16,000 × g (14,000 rpm) to concentrate your DNA.
?. [Gel-based Size Selection and Purification]
Discard the SV Minicolumn, and store the microcentrifuge tube containing the eluted DNA at or .
The volume of the eluted DNA should be approximately 18-19 μl.