Protocol Citation: eaglefishgeneticslab 2023. RADSeq protocol EFGL. protocols.io https://dx.doi.org/10.17504/protocols.io.kqdg39bjqg25/v1
Manuscript citation:
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: February 17, 2023
Last Modified: June 07, 2023
Protocol Integer ID: 77120
Keywords: Massively parallel sequencing, MPS, restriction-site associated DNA, RAD, sequence capture, genotyping, population genetics, Eagle Genetics Lab, EFGL, Radseq, radseq protocol efgl modified, radseq protocol efgl modified from protocol, protocol
Abstract
Modified from protocol of Sean O’Rourke and Mike Miller published in:
Citation
LINK
Materials
MATERIALS
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S
PstI - 10,000 unitsNew England BiolabsCatalog #R0140S
BfaI - 500 unitsNew England BiolabsCatalog #R0568S
NEBNext Ultra II DNA Library Prep Kit for Illumina - 24 rxnsNew England BiolabsCatalog #E7645S
Restriction enzymes:
PstI: NEB
BfaI-HF: NEB
CutSmart buffer
BestRAD plate adaptors:
BestRAD adaptors allow the addition of inline barcodes, and the isolation of RAD tags through purification by and enzymatic liberation of fragments from streptavidin beads. Well-specific Hamming barcodes (septamers in this case) are specified in an Excel spreadsheet and are not actually ordered as N’s. Top oligos are 5’-biotinylated and contain the 3’ PstI/SbfI overhang. Bottom oligos are 5’-phosphorylated to promote ligation.
Top oligo
Anatomy: biotin spacer SbfI barcode overhang
Sequence: /5Biosg/GTACGT CCTGCAGG NNNNNNN TGCA
Bottom oligo
Sequence: /5Phos/NNNNNNN CCTGCAGG ACGTAC
Ordering Specs
- Standard plate oligos
- 25 nmol standard desalting
- dry
- $1859.52 total for a set of 48
Streptavidin purification:
Dynabead M280 strepatividin, 2mL (GrizMart, ~ $500)
2X Binding and Wash Buffer:
1. 10 mM TrisHCl (pH 7.5)
2. 1 mM EDTA pH 8.0
3. 2 M NaCl
4. Concentrated (or dry) stocks should be available in chemical cabinet.
NextGen library prep:
NEBNext Ultra II
Kit
- includes reagents for end-repair, A-tail, ligation
- NEB E7645S 24 rxns, GrizMart, Fisher
- ~ $590
Oligos (12-plex)
- Indexed oligos containing Illumina sequencing primer sequences and required for annealing to flow cell. Added via PCR to NEBNext adaptor-ligated fragments.
- 12 barcoded i7 indexing primers
- 1 universal (i5) oligo
- NEBNext adaptor w/ USER enzyme
- NEB E7335S
- Grizmart
- ~$110
Universal primer with molecular barcode
- Modeled after i5 index primers from NEB #E7600 (p21 of manual)
- N’s specify the equimolar addition of dATP, dTTP, dGTP, and dCTP during synthesis
• Not truly random. Some GC-bias in addition
• Should be sufficiently diverse to detect PCR duplicates
- Used in place of the universal i5 oligo in NEB #E7335S
- IDT, HPLC-cleaned - $90.50
- Primer with molecular barcode in bold:
5'-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACN NNN NNN NAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T-3’
- Compare to NEBNext i506 primer with indexing barcode in bold:
5´-AAT GAT ACG GCG ACC ACC GAG ATC TAC ACT AAT CTT AAC ACT CTT TCC CTA CAC GAC GCT CTT CCG ATC*T-3´
Reaction purification and size selection:
- Ampure XP magnetic beads
- Polyethylene glycol (PEG-8000)
- NaCl
- Nice summary as of Dec 2017: http://core-genomics.blogspot.com/2012/04/how-do-spribeads-work.html
Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warings and hazard information.
Annealing TOP/BOTTOM BestRAD adapters
45m
Preparing BestRAD adapters (Skip section if BestRAD adapters have been previously annealed)
Note
We have used 3 restriction enzymes with 6 base pair recognition sites (PstI, BamHI, and HindIII) and 1 enzyme with an 8 bp recognition site (SbfI) in our protocol. Adapters should be compatible with the base pair overhang that each restriction enzyme leaves. Both PstI and SbfI leave the same overhangs so the same adapters can be used with either enzyme. Adapters are ordered from Integrated DNA Technologies as single stranded top adapters in one plate and single stranded bottom adapters in another plate. They have to be re-hydrated, diluted to the proper concentration, and annealed prior to beginning the BestRAD protocol. See attached documents below for the sequences of our top and bottom adapters that we use. Note that the file format is for importing into IDTdna's Custom DNA Oligo, single-stranded DNA, 96 well plate form (https://www.idtdna.com/pages/products/custom-dna-rna/dna-oligos/custom-dna-oligos). We've ordered adapters a variety of ways, but the best/easiest is to order the adapters at 25nmole scale, standard desalting purification, PCR plate type, dry ship option, normalized yield and nmol quantity of 10. All top adapters are biotinylated.
We typically make a 1µM working concentration of annealed adapters if the restriction enzyme we are using is a 6 base-pair cutter. We make a 50nM working concentration of annealed adapters if the restriction enzyme is an 8 base-pair cutter. A 6 bp cutter will cut more frequently so we need a higher concentration of it.
BamHI_adapter_bottom.txt BamHI_adapter_top.txt HindIII_adapter_top.txt HindIII_adapter_bottom.txt Sbf_Pst_adapter_top.txt Sbf_Pst_adapter_bottom.txt Note
If you already have a plate of diluted, annealed adapters, skip to Step 5
Equipment and supplies needed for steps 1-4:
thermalcycler (We have several different types of thermalcyclers. Here's an example of one we use often)
Equipment
Mastercycler Pro
NAME
thermalcycler
TYPE
Eppendorf
BRAND
No Longer Manufactured
SKU
Equipment
EPPENDORF SCIENTIFIC CENTRIFUGE 5804
NAME
plate centrifuge
TYPE
Eppendorf
BRAND
02-262-8153PM; discontinued
SKU
Equipment
Repeater-M4
NAME
pipette
TYPE
Eppendorf
BRAND
14-287-150
SKU
Equipment
ALPS 25 V
NAME
manual heated plate sealer
TYPE
Thermo Scientific
BRAND
AB-0384/110; discontinued
SKU
p10 multi-channel pipette and tips
benchtop centrifuge
1 mL Eppendorf combi-tips (Fisher Scientific; 13-683-703)
vortex
heat seals (Fisher Scientific; AB-0745)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
Reagents needed:
Bottom adapter plate
Top adapter plate
Duplex BufferIDTCatalog #11-01-03-01
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Making a 100µM stock of bottom and top adapters
Assuming the bottom and top adapters were ordered dry and at a 25 nmole scale and normalized to 10nmol, first re-hydrate to 100µM by adding 100 µL of nuclease-free water to each well with repeater pipette and combitip
Heat seal the plate
Expected result
Now you should have a plate of 100 µL top adapters at 100µM concentration and a plate of 100 µL bottom adapters at 100µM concentration.
Making a 10uM stock of bottom and top adapters
Note
Mix the 100µM adapter plates by gently vortexing, then briefly spin the plates down
Use a p10 multi-channel pipette to transfer 10 µL of each top adapter to a new plate.
Using a repeater pipette and combitip, add 90 µL of nuclease-free water to the same wells of the new plate.
Heat seal the plate
Note
Even though you need to use the plates immediately in the next steps, you still need to heat seal them after every dilution so you can vortex the plate and spin it down
Repeat for the bottom adapters from step 1.4.
Heat seal the plate
Expected result
Now you should have a plate of 100 µL top adapters at 10µM concentration and a plate of 100 µL bottom adapters at 10µM concentration.
Choose between the following options:
- If using 6 base pair cutter (PstI, BamHI, HindIII), need to make 1µM dilution of BestRAD adapters
- If using 8 base pair cutter (SbfI), need to make 50nM dilution of BestRAD adapters
6 base pair cutter, 1µM adaptersFrom 22 to 212 steps
Making a 1.0µM working concentration of COMBINED bottom and top adapters
Note
Mix the 10µM adapter plates by gently vortexing, then briefly spin the plates down
Using a multi-channel pipette, add 2.5 µL of the 10µM top adapters to a new plate. Discard tips.
Using a multi-channel pipette, add2.5 µL of the corresponding 10µM bottom adapters (the same well position as the top adapter plate) to the same well of the new plate. Discard tips.
Mix the duplex buffer by vortexing, then briefly spin down
Using a repeat pipette and a 1mL combi-tip, add 45 µL of duplex buffer to all wells.
Heat seal the plate
Expected result
Now you have a single plate with 50 µL of bottom and top adapters combined at 1µM concentration.
Annealing adapters
Run the plate of COMBINED adapters (50 µL ) on a thermalcycler using the following program (In our lab, the program can be found using the menu options General -> RAD -> duplexAssay)
CHECK THE PROGRAM TO MAKE SURE IT'S CORRECT
- 95 °C for 00:02:00
- Reduce temperature by 1 °C for 00:01:00
- repeat until temperature is down to 4 °C
Expected result
Now you have a single plate with 50 µL of bottom and top adapters combined at either 1µM concentration if you were following the 6 bp cutter steps or 50nM concentration if you were following the 8 bp cutter steps. These BestRad adapters include the barcodes for each sample
3m
Check to see if there's sufficient stock made of 2X B+W buffer (needed for step 15, size selection) and stored in 50mL conical tube at room temperature. If not, make it
Note
Equipment and supplies needed for this step:
50mL conical tube (Fisher Scientific; 12-565-271)
Reagents needed:
Tris-HCl 1.0M pH 7.5Fisher ScientificCatalog #AAJ60636K2
EDTA 0.5M pH 8.0Fisher ScientificCatalog #AAJ62786AP
NaClFisher ScientificCatalog #BP358-1
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Add the following to a 50mL conical tube:
- 500 µL 1M Tris-HCl pH 7.5
- 100 µL 0.5M EDTA pH 8.0
- 5.844 g NaCl
- Add enough water to obtain 50 mL final volume
Note
Final concentration is 2X: 10mM Tris-HCl pH 7.5, 1mM EDTA pH 8.0, 2M NaCl
Choose from the following options:
Quantification of <48 samples
Quantification of >48 samples
<48 Samples202 steps
DNA quantification <48 samples
Quantify DNA with Qubit
Note
Note that for sample sizes less than 48ish, we quantify each sample individually using a Qubit fluorometer. Otherwise, we use a plate reader.
Note
Equipment and supplies needed for this step:
Equipment
Qubit Fluorometer
NAME
Fluorometer to quantify DNA
TYPE
Invitrogen
BRAND
Q33226
SKU
Qubit assay tubes (Fisher Scientific; Q32856)
Eppendorf 2mL Snap-Cap Microcentrifuge Safe-Loc Tubes (Fisher Scientific; 05-402-7)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
p10 pipette and tips
p200 pipette and tips
Reagents needed:
DNA tray(s)
Qubit dsDNA HS assay kitFisher ScientificCatalog #Q32854
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36 if dilution necessary
Enter sample data info into “sampleData” tab of BestRAD_library_prep_labData v1.2.xlsx (attached in the DESCRIPTION tab of this protocol. It has example data for 20 samples.)
Qubit all samples with Qubit dsDNA HS assay kit.
Follow the manufacturer's protocol to make a working solution (1:200) of dye https://tools.thermofisher.com/content/sfs/manuals/Qubit_dsDNA_HS_Assay_UG.pdf
Make sure you set the sample volume and the output to be read in ng/µl, not ng/mL
Take 2 readings per sample and enter them into columns F and G in "qubitData" tab in BestRAD_library_prep_labData v1.2.xlsx. The spreadsheet has a column that will take the average. The target is 10ng/µl.
If DNA concentration is off the Qubit scale,
- perform a 1:5 dilution (1 µL of DNA, 4 µL of water) in a new PCR plate
- Re-Qubit diluted samples
If DNA concentration is too low (less than 9ng/µl), reach out to lead biologist for replacement samples, or re-extract DNA then re-Qubit new samples
DNA Normalization
Normalize DNA to 10ng/µl
Note
Equipment and supplies needed for this step:
manual heated plate sealer
plate seals (Fisher Scientific; AB-0745)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
p10 pipette and tips
p200 pipette and tips
vortex
plate centrifuge
Results from "Norm" tab of BestRAD_library_prep_labData v1.2.xlsx
Reagents needed:
DNA tray(s)
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
The table in “Norm” tab of BestRAD_library_prep_labData v1.2.xlsx has been formulated to normalize all samples to 10ng/µl at a 30 µL final volume, and you’ll need 10 µL for each sample in next step.
Label a new PCR plate: Normalized DNA for [insert RADseq project name], date, initials
Pipette the amount of DNA indicated in column F of "Norm" tab of BestRAD_library_prep_labData v1.2.xlsx from the DNA tray to corresponding well position in new Normalization plate
Pipette the amount of nuclease-free water indicated in column G of "Norm" tab to corresponding well position in new Normalization plate
Heat seal plate, Mix by gently vortexing, then briefly spin the plate down
Expected result
Tray(s) with 30 µL of your samples normalized to 10ng/µl
Store at 4 °C if you plan to start the library in the next couple of days,
otherwise -20 °C
Day 1 - Digestion
Digest the normalized DNA with a restriction enzyme
Note
Equipment and supplies needed for this step:
thermal cycler
manual heated plate sealer
plate seals (Fisher Scientific; AB-0745)
p10 pipette and multi-channel pipette and tips
p200 pipette and tips
Repeater-M4 pipette
0.1mL combi-tip (Fisher Scientific; 13-683-700)
unskirted 96 well PCR trays (Fisher Scientific; AB-0700)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-791)
vortex
plate centrifuge
timer
Reagents needed:
10µl of normalized DNA per sample
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
1 of the following restriction enzymes
PstI - 50,000 unitsNew England BiolabsCatalog #R0140L
SbfI-HF - 2,500 unitsNew England BiolabsCatalog #R3642L
BamHI - 10,000 unitsNew England BiolabsCatalog #R0136S
HindIII - 10,000 unitsNew England BiolabsCatalog #R0104S
NOTE: All restriction enzymes' concentrations are 20,000U/µl
Thaw reagents to prep for master mix
- Whichever restriction enzyme chosen for the project (PstI, SbfI, BamHI, or HindIII)
- CutSmart Buffer 10X
- Normalized DNA if it's been in the freezer
Add reagents from table below to a 1.5mL Lo-Bind tube to make a master mix. Add 5-10 to your sample size to account for pipette error. For example, if you have 20 samples, multiply the volumes per sample by 25 .
Note
Recipe above assumes 20units/µl of enzyme and 2.4 units per reaction
Vortex and briefly spin down
Label PCR plate "Digestion"
Using a p10 pipette or 0.1mL combitip, add 2 µL of master mix to each well
Using a multi-channel p10 pipette, add 10 µL of normalized DNA to corresponding wells
Heat seal PCR plate, vortex gently and quickly spin down.
Run the following thermalcycler program (In our lab, the program is "1 RE-digest" and can be found using the menu options General -> RAD)
- 37 °C for 01:00:00
- 85 °C for 00:30:00
CHECK THE PROGRAM TO MAKE SURE IT'S CORRECT
1h 30m
Set a timer for 01:15:00
Prep next step, about 00:15:00 prior to end of thermalcycler program
Expected result
Tray labeled "Digestion" with 12 µL of ~100 ng of DNA per sample that has been digested with a restriction enzyme
1h 30m
Day 1 - Ligation
Ligate adapters onto digested DNA
Note
Equipment and supplies needed for this step:
thermal cycler
plate centrifuge
manual heated plate sealer
plate seals (Fisher Scientific; AB-0745)
p10 pipette and multi-channel pipette and tips
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-791)
vortex
Reagents needed:
plate of annealed adapters
plate of digested DNA
CutSmart Buffer 10X (comes with restriction enzyme)New England Biolabs
100mM ATP SolutionThermofisherCatalog #R0441
T4 ligaseNew England BiolabsCatalog #M0202M
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
- Thaw BestRAD adaptors (prepared in steps 1-4) and reagents for Ligation Master Mix (listed above)
gently vortex and spin down all reagents
Make Ligation master mix in a 1.5mL Lo-Bind tube by combining reagents below. Multiply by the number of samples you have + 5-10 extra to account for pipette error.
Remove Digestion PCR plate from thermocycler (Step 9.9), quick spin
With a multi-channel p10 pipette, add 2 µL of annealed adaptors to each well in Digestion PCR plate, corresponding to the same well setup in the "library prep" tab of BestRAD_library_prep_labData v1.2.xlsx. Discard tip after each use.
Using a p10 pipette, add 2 µL of Ligation master mix to each well. Discard tip after each use.
Heat seal PCR plate, gently vortex, quick spin
Overnight
Run thermalcycler program: (In our lab, the program is called "2 Ligation" and can be found using the menu options : General -> RAD)
- 20 °C for 16:00:00
- 80 °C for 00:30:00
Note
Do not let plate sit at 4°C hold for too long, recommended to proceed to next step as soon as program ends.
Expected result
16 µL of digested DNA per sample with BestRad adapters attached
16h 30m
Day 2 - Sonication
Prepping the Sonicator
Note
Equipment and supplies needed for this step:
Equipment
QSonica SONICATOR SYSTEM Q800R1
NAME
Sonicator
TYPE
QSonica
BRAND
discontinued
SKU
Two 1 Liter bottles for de-ionized water
Reagents needed:
de-ionized water (From Fish Health Lab)
Fetch ~2 L of de-ionized water from the Fish Health Lab. Take containers with you
Connect all corresponding tubes to the machine as labeled and screw on the filter
Empty 1 L of de-ionized water into sonicator and start the machine.
When the filter is fully saturated, and water cycle is complete, add more de-ionized water until the level is just below the top white spout.
Proceed to next step, and let machine cool to4 °C
Day 2 - Pool samples
Note
Equipment and supplies needed for this step:
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-791)
OR
2.0mL Lo-Bind microcentrifuge tube (Fisher Scientific; 13-698-792)
Reagents needed:
"Digestion" plate of digested samples with BestRad adapters attached
Pooling samples
- Using a p200 single channel pipette, pool all 16 µL of each well from Digestion PCR plate into a new 1.5mL Lo-Bind tube
- If library contains more than 93 samples, pool into a 2.0mL Lo-Bind tube instead
Expected result
A library tube with pooled samples, volume = 16 µL X number of samples
Day 2 - Sonicate, precipitate, ligate library
purifying pooled library
Note
Equipment and supplies needed for this step:
Equipment
DynaMag Spin
NAME
Magnet for microcentrifuge tubes
TYPE
Invitrogen
BRAND
12320D
SKU
p200 pipette and tips
p1000 pipette and tips
1.5mL Lo-Bind microcentrifuge tubes (Fisher Scientific; 13-698-791)
Bioruptor Plus TPX microtubes (Fisher Scientific; C30010010-50)
5.0mL Axygen Polypropylene Snaplock non-sterile tube (Fisher Scientific; 14-568-100)
OR
15mL conical tube (Fisher Scientific; 14-959-53A)
vortex
benchtop centrifuge
Reagents needed:
tube with pooled library
Ampure XP beadsBeckman CoulterCatalog #A63881
low TEFisher ScientificCatalog #AAJ75793AP
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
100% ethanol (laboratory grade, non-denatured!)
Note
Ampure XP beads have been aliquoted into 2 mL tubes to prevent contamination of the stock. In general, whenever they are used in the protocol, they should be vortexed thoroughly to ensure mixing
Note
if library contains >18 samples
- Rule of thumb: Each tube should have 200-300μl of pooled samples to allow adequate room for 80% ethanol wash step
- At the end, the combined total low TE volume of all tubes must be 210 µL
Using the table below, calculate the respective volumes required, or enter in number samples in "Pooling_calculator" tab in library prep spreadsheet to have volumes calculated
E.g. If N=48, then you need 3 tubes, the volume of pooled samples =256 µL , volume of AMPure XP beads = 256 µL , volume of 80% EtOH = 1.024 mL , volume of low TE = 70 µL
Determine how many 1.5mL Lo-Bind tubes you need based on table above.
Aliquot the pooled library into the tubes equally
Add an equal volume of AMPure XP beads to pooled library amount in a 1:1 ratio
Gentle vortex, quick spin.
Incubate tube(s) at room temperature for 00:10:00
10m
During the 10 min incubation, prep 80% ethanol in 5mL tube(s) or 15ml conical
Note
e.g. to make 5 mL of 80%, mix 4 mL of 100% ethanol and 1 mL of nuclease-free water
For 100 samples: need 12 mL 100% ethanol, 3 mL water
For 74 samples: need 9.6 mL 100% ethanol, 2.4 mL water
For 56 samples: need 8 mL 100% ethanol, 2 mL water
For 37 samples: need 5.6 mL 100% ethanol, 1.4 mL water
The volumes above take into account the extra ~2mL of 80% ethanol needed in the rest of the protocol
Place tube(s) on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube(s) for 00:05:00
5m
While the tube(s) are still on the magnet, using a p200 pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard supernatant
While tube(s) are still on the magnet, add 2X total volume of 80% ethanol to the beads and incubate for 00:00:30 .
Note
e.g. If 48 samples: 256 µL of library in 3 tubes: 2X total volume = (256 µL pooled library + 256 µL beads)*2 = 1024 µL 80% ethanol per tube
30s
Discard supernatant as in step 13.8
repeat step 13.9 one more time
Discard supernatant as in step 13.8
Leave tube(s) on magnet and allow any residual ethanol to evaporate by letting stand for 00:05:00 uncovered
5m
Take tube(s) off the magnet
Add low TE to each tube, refer to table at the beginning of this section for how much
Pipette up and down a few times to completely resuspend the bead pellet
Incubate tubes(s) at room temperature for 00:05:00 .
5m
Place tube(s) in magnetic stand for 00:05:00
Note
THE SUPERNATANT HAS THE DNA IN IT NOW. YOU WANT TO KEEP THE SUPERNATANT
5m
Using a p200 pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove the supernatant and put into a TPX tube.
Repeat for all library tubes on the magnet. Deposit all supernatants into same TPX tube
Pipette210 µL of water into another TPX tube
LABEL BOTH TPX TUBES TO AVOID MIXUP!!
Expected result
1 TPX tube with 210 µL of pooled, digested DNA fragments
1 TPX tube with 210 µL of water to use as a balance in the sonicator
Sonication
Note
Equipment and supplies needed for this step:
Sonicator (prepped in step 7)
Reagents needed:
TPX tube with library
TPX tube with water as balance
Sonicate library for 00:04:30 at 20% capacity, and at 4 °C
4m 30s
Remove tubes after sonication has completed
OK to sit in room temperature for next step.
Discard TPX vial containing water
Turn off sonicator and coolant pump
Expected result
210 µL of pooled, sonicated library in tube.
Note
DNA fragments should be between 200-500bp long. Can check this on gel or Bioanalyzer here if troubleshooting needed
Some fragments now lack BestRad adapters after sonication. The next step gets rid of these
Day 2 - Bead Selection
Prep buffers for bead binding in Step 17
Note
Equipment and supplies needed for this step:
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
p200 pipette and tips
p1000 pipette and tips
heat block
Reagents needed:
2X B+W buffer (directions on how to prepare in step 5)
CutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Thaw 10X CutSmart Buffer at room temperature
Check heat block to make sure it's on and set to56 °C
To a 1.5mL Lo-Bind tube add:
- 25 µL 10X CutSmart Buffer
- 225 µL water
To a 1.5mL Lo-Bind tube add:
- 250 µL 2X B+W buffer
- 250 µL water
To a 1.5mL Lo-Bind tube add:
- 175 µL 2X B+W buffer
- 175 µL water
Place tube from step 15.5 in heat block set to 56 °C
Expected result
1 tube of 250 µL 1X CutSmart Buffer
1 tube of 500 µL 1X B +W Buffer
1 tube of warm 350 µL 1X B +W Buffer in heat block
Prep Dynabead M280 streptavidin magnetic beads
Note
Equipment and supplies needed for this step:
p200 pipette and tips
vortex
benchtop centrifuge
DynaMag Spin
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
2X B+W buffer made in Step 5
Dynabeads M-280 StreptavidinFisher ScientificCatalog #11-206-D
Vortex stock bottle of Dynabeads and transfer 20 µL to new 1.5mL Lo-Bind tube
Note
Dynabeads are temperature sensitive, place beads back in fridge immediately after use
Place tube on the DynaMag Spin for 00:00:10
10s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 20 µL supernatant
Remove tube from magnet and add 100 µL 2X B+W buffer
Gently vortex for 00:00:30 , then quick spin
30s
Place tube on the magnet for 00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 100 µL supernatant
Repeat steps 16.4 through 16.7
Take tube off magnet, re-suspend beads in 200 µL of 2X B+W buffer
Vortex
Expected result
200 µL of Dynabead M280 Streptavidin beads prepped for binding
Day 2 - binding DNA to beads
22m
Bind DNA to beads
Note
Equipment and supplies needed for this step:
p1000 pipette and tips
p200 pipette and tips
benchtop centrifuge
DynaMag Spin
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
sonicated library from Step 14.2
prepped beads from 16.9
2X B+W buffer made in Step 5
1X B+W buffer from Step 15.4
warm 1X B+W buffer from Step 15.6
Transfer all 210 µL of sonicated library to freshly prepped 200 µL Dynabeads tube from Step 16.9
Incubate for 00:20:00 at room temperature while gently vortexing every 2 minutes for 10 seconds (e.g 20:00 minute mark, vortex till 19:50. At 18 minutes, vortex till 17:50, etc)
20m
Quick spin then place tube on the DynaMag Spin for 00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 410 µL supernatant
Take tube off magnet, re-suspend beads in 150 µL of 1X B+W buffer from Step 15.4
Mix by pipetting
Place tube on the DynaMag Spin for 00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 150 µL supernatant
Repeat steps 17.5 - 17.7 2 more times
Take tube off magnet, re-suspend beads in 150 µL of warm 1X B+W buffer from Step 15.6
Mix by pipetting
Note
For following steps, keep warm 1X B+W buffer in heat block at 56 °C when not in use
Place tube on the DynaMag Spin for 00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 150 µL supernatant
Repeat steps 17.9 - 17.11 1 more time
Take tube off magnet, re-suspend beads in 200 µL of 2X B+W buffer
Mix by pipetting
Expected result
A tube with 200 µL of DNA fragments with BestRad adapters bound to the beads.
Liberate DNA from beads
Note
Equipment and supplies needed for this step:
thermal cycler
p10 pipette and tips
p200 pipette and tips
p1000 pipette and tips
plate centrifuge
DynaMag Spin
magnet stand for plate
unskirted PCR plate (Fisher Scientific; AB-0700)
manual heated plate sealer
plate seal (Fisher Scientific, AB-0745)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
DNA and beads from Step 17.13
1X CutSmart buffer from Step 15.3
SbfI-HF - 2,500 unitsNew England BiolabsCatalog #R3642L
Re-suspend beads in 100 µL of 1X CutSmart buffer from Step 15.3
Mix by pipetting
Place tube on the DynaMag Spin for 00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 300 µL supernatant
Repeat steps 18.1 - 18.3. Note that this time around the volume of the supernatant to remove will be 100 µL
Take tube off magnet, re-suspend beads with bound DNA in 40 µL 1X CutSmart
Mix by pipetting
Transfer all 40 µL beads mix into well C3 of PCR plate
Note
In our lab, we don't use any PCR tubes, only plates. This step can be done in a tube that would fit a thermalcycler.
Pipette 2 µL SbfI-HF (NEB R3642L) into the well
Use a p200 pipette set to 20μl to mix the solution
DO NOT VORTEX or SPIN DOWN PLATE
Heat seal PCR plate
Run thermalcycler program: 3 37hold (General -> RAD -> 3 37hold)
- 37 °C for 01:00:00
1h
After program has completed, give the plate a quick spin
Place plate on magnet in plate format for 00:00:30
Note
In our lab, we rigged a magnet to work on a plate. However, there are commercially available plate magnets. If you performed the incubation in a PCR tube, transfer the liquid to a tube that will fit the tube magnet stand.
30s
transfer all 42 µL supernatant to new 1.5mL Lo-Bind tube
Expected result
42 µL of DNA fragments with BestRad adapters attached in tube
Size select DNA fragments
Note
Equipment and supplies needed for this step:
p200 pipette and tips
DynaMag Spin
unskirted PCR plate (Fisher Scientific; AB-0700)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
Reagents needed:
80% ethanol from Step 13.6
DNA and beads from Step 18.13
low TEFisher ScientificCatalog #AAJ75793AP
Agencourt AMPure XPBeckman CoulterCatalog #A63880
Add 42 µL of AMPure XP beads to 42 µL DNA tube
Mix by pipetting
Incubate 84 µL mixture at room temperature for 00:10:00
10m
Place tube on the DynaMag Spin for 00:05:00
5m
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 84 µL supernatant
While still on magnet, add 168 µL of 80% ethanol (made the same day) to beads and incubate for 00:00:30
30s
While the tube is still on the magnet, using a pipette and tip, insert the tip into the tube away from the bead pellet and slowly remove and discard the 168 µL supernatant
Repeat wash steps 19.5 - 19.6
Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for 00:05:00 uncapped
5m
Resuspend beads with 55 µL low TE
Mix by pipetting
Incubate at room temperature for 00:05:00
5m
Place on DynaMag Spin for 00:05:00
Note
NOW THE SUPERNATANT HAS THE DNA IN IT
5m
Transfer 55 µL supernatant to PCR plate (well C3 is preferred)
Note
If stopping is necessary, transfer supernatant to a new 1.5mL Lo-Bind tube and store in -20 °C
Expected result
55 µL purified DNA fragments with BestRad adapters attached
Day 2 - Ligation
Ligating NEBNext adapters to DNA fragment
Note
Equipment and supplies needed for this step:
thermal cycler
p10 pipette and tips
p200 pipette and tips
manual heated plate sealer
plate seals (Fisher Scientific, AB-0745)
plate centrifuge
vortex
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
timer
Reagents needed:
NEBNext Ultra II DNA Library Prep Kit for Illumina - 96 rxnsNew England BiolabsCatalog #E7645L
1M Tris-HCl pH7.5Fisher ScientificCatalog #AAJ60636K2
purified DNA from Step 19.12
Note
The protocol calls for 10mM Tris-HCl pH 7.5, so make a dilution if needed
The NEBNext kit has 2 components: End Prep reagents and Ligation reagents
Thaw reagents from NEBNext End prep kit
- Ultra II End Prep Enzyme Mix
- Ultra II EP Buffer
Add 3 µL of Ultra II End Prep Enzyme Mix and 6.5 µL of Ultra II EP Buffer to the well of the tray with the 55 µL of DNA
Mix by pipetting (set p200 to 40µl)
Heat seal the plate and quick spin
Run thermalcycler program: 4 End Prep (General -> RAD -> End Prep)
- 20 °C for 00:30:00
- 65 °C for 00:30:00
Set a timer for 45 min
1h
While program is running, drain sonicator.
1. Disconnect the tube labeled “coolant supply” from the sonicator and remove the white nozzle end completely.
2. Turn on the pump, and tip the sonicator backwards to drain as much water as possible, then turn pump off.
3. Use paper towels to absorb all remaining water in sonicator
4. Leave lid open for a day or 2 to air dry. Unscrew the filter, empty out any remaining water and let it sit to air dry.
When timer goes off, thaw the following reagents for ligation:
- Ultra Prep II Ligation master mix
- NEBNext Adaptor
- Ligation Enhancer
Dilute NEBNext adapter 1:10 in a new 1.5mL Lo-Bind tube
1 µL of NEBNext Adaptor + 9 µL 10mM Tris HCl pH 7.5
Note
The manufacturer's protocol says to dilute the NEBnext adapter IF the DNA input is ≤ 100 ng. We don't check this and assume the adapter always needs diluting. Also, the protocol says to mix the 10mM Tris HCl pH 7.5 with 10mM NaCl, but we haven't been doing this (May, 2023).
Make Ligation master mix in a 1.5mL Lo-Bind tube by combining reagents below. Multiply by the number of samples you have + 5-10 extra to account for pipette error.
Note
The NEBNext adapter is a double stranded hairpin that will ligate to both sides of the DNA fragments. It contains the priming sites for the PCR primers in Step ______
Add master mix to well of plate with DNA (the volume should be 83 µL )
Mix by pipetting (set p200 to 40µl and mix)
Heat seal the plate, quick spin
Run thermal cycler program: 5 Adaptor Ligation (General -> RAD -> 5 Adapter Ligation
20 °C for 00:15:00
15m
After program has ended add 3 µL of NEBNext USER enzyme
Mix by pipetting as above
Note
The NEBNext adapter has a uracil base in it. The USER enzyme cleaves the hairpin adapter at the uracil so now the DNA fragments are linear and have the PCR priming sites on both sides needed in Step 22
Heat seal the plate, quick spin
Run thermalcycler program: 6 USER enzyme (General -> RAD -> 6 USER enzyme)
37 °C for 00:15:00
Expected result
A well in a PCR plate with 86 µL of DNA fragments that have BestRad adapters on one side with the barcodes and NEBNext adapters on both sides with the PCR priming sites
15m
Day 2 - Ampure Size Selection and Clean-Up
20m
Size selection and cleanup of adaptor-ligated DNA
Note
Equipment and supplies needed for this step:
p10 pipette and tips
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tubes (Fisher Scientific, 13-698-791)
DynaMag Spin
unskirted 96 well PCR tray (Fisher Scientific; AB-0700)
Reagents needed:
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Agencourt AMPure XPBeckman CoulterCatalog #A63880
low TEFisher ScientificCatalog #AAJ75793AP
80% ethanol
Transfer all 86 µL solution from 96-well unskirted PCR plate to a new 1.5mL Lo-Bind tube
Add 13.5 µL of nucelase-free water and 44 µL AMPure XP beads
mix by pipetting
Incubate at room temperature for 00:05:00
5m
Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for 00:05:00
5m
Transfer all 143.5 µL supernatant to new 1.5mL Lo-Bind tube
Add 26 µL AMPure XP beads
mix by pipetting
Incubate at room temperature for 00:05:00
5m
Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for00:05:00
5m
Remove and discard supernatant (~170 µL )
While still on magnet, add 200 µL of fresh 80% ethanol to beads and incubate for 00:00:30
30s
Repeat 21.9 - 21.10
Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for 00:05:00 uncovered
5m
Take the tube off the magnet and re-suspend the beads in 17 µL low TE
Mix by pipetting
Incubate for 00:02:00 at room temperature
2m
Put tube on DynaMag Spin for 00:05:00
5m
Transfer 15 µL into a new unskirted PCR plate
OR
If stopping for the day, transfer into a new 1.5mL Lo-Bind tube, store at -20 °C
Expected result
15 µL of purified DNA fragments with BestRAD and NEBNext adapters
PCR amplification
Note
Equipment and supplies needed for this step:
thermal cycler
p10 pipette and tips
200 pipette and tips
manual heated plate sealer
plate seals (Fisher Scientific, AB-0745)
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
vortex
plate centrifuge
Reagents needed:
NEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S
15 µL of DNA with ligated adapters from Step 21.16
Thaw reagents from NEBNext Multiplex Oligo kit (orange box) labeled: “RADseq PCR Amp”
Select an Index (i7) from the NEBNext Multiplex Oligo kit to use and record it in the "Final Library" tab in BestRAD_library_prep_labData v1.2.xlsx.
Note
The i7 MUST BE unique per library. If you are preparing more that 1 library, use different i7s.
Add the following reagents to the 15 µL of DNA:
Total volume is 50 µL
Heat seal the plate, gently vortex, quick spin
Run thermal cycler program: General -> RAD-> 7 NEBNext PCR 11x
Runs for about 00:28:00
Note
The BestRAD protocol says to perform 4-12 cycles of PCR. Currently we use 11.
28m
Transfer 50 µL PCR product to new 1.5mL Lo-Bind tube
Note
Do NOT stop here. Complete PCR purification below
PCR purification
Note
Equipment and supplies needed for this step:
p200 pipette and tips
1.5mL Lo-Bind microcentrifuge tube (Fisher Scientific, 13-698-791)
DynaMag Spin
vortex
Reagents needed:
Agencourt AMPure XPBeckman CoulterCatalog #A63880
low TEFisher ScientificCatalog #AAJ75793AP
80% ethanol
Add 45 µL AMPure XP beads to the 50 µL of DNA from Step 22.6
mix by pipetting
Incubate at room temperature for 00:05:00
Place tube on DynaMag Spin, and allow the magnetic beads to stick to the side of the tube for00:05:00
Remove and discard supernatant (~95 µL )
While still on magnet, add 200 µL of fresh 80% ethanol to beads and incubate for 00:00:30
Repeat 21.9 - 21.10
Leave tube on magnet and allow any residual ethanol to evaporate by letting stand for 00:05:00 uncovered
Take the tube off the magnet and re-suspend the beads in 33 µL low TE
Mix by pipetting
Incubate for 00:02:00 at room temperature
Put tube on DynaMag Spin for 00:05:00
Transfer 28 µL into a new 1.5mL Lo-Bind tube
Label the tube with species "Radseq", library number, date
Store at -20 °C if stopping for the day
Expected result
28 µL of final, undiluted library
Day 3 - quantify library
1h 3m
Qubit Rad library
Note
Equipment and supplies needed for this step:
Qubit fluorometer
Qubit assay tubes (Fisher Scientific, Q32856)
1.5mL or 2.0mL microcentrifuge tube
p10 pipette and tips
p200 pipette and tips
vortex
benchtop centrifuge
Reagents needed:
DNA library from Step 23.11
Qubit dsDNA HS assay kitFisher ScientificCatalog #Q32854
Thaw library to room temperature
Make 1:200 diluted dye mix in a microcentrifuge tube
199 µL Qubit buffer
1 µL Qubit dye
Pipette 198 µL of diluted dye mix from Step 24.2 and 2 µL of the library into a qubit assay tube
Vortex thoroughly, quick spin
Incubate at room temperature for 00:03:00
3m
Put qubit assay tube in Qubit Fluorometer and take 2 readings per sample and enter them into BestRAD_library_prep_labData v1.1 spreadsheet in “Final Library” tab. The spreadsheet has a column that will take the average. The target is 4-20 ng/µl.
Note
Don't use any tube other than the qubit assay tubes in the Qubit.
Make sure you set the sample volume and the output to be read in ng/µl, not ng/mL
Expected result
26 µL of your Radseq library at a concentration
E-gel of library
Note
Equipment needed
p10 pipette and tips
p200 pipette and tips
parafilm
vortex
benchtop centrifuge
timer
thumb drive
Equipment
E-Gel Power Snap Electrophoresis System
NAME
e-gel electrophoresis
TYPE
Invitrogen
BRAND
G8300
SKU
Reagents needed
Invitrogen E-Gel EX Agarose Gels 1%Fisher ScientificCatalog #G402021
Invitrogen E-Gel 1 Kb Plus Express DNA ladderFisher ScientificCatalog #10-488-091
6X loading dye (comes with ladder)
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
Thaw 1 Kb ladder, loading dye, and RAD library if needed
Gentle vortex, quick spin
Cut strip of parafilm, tape to benchtop if necessary
Onto parafilm, pipette 15 µL of 1 Kb ladder. Add5 µL of nuclease free water to the ladder. Mix by pipetting
Onto parafilm, pipette 5 µL of the RAD library. Add 3 µL of loading dye and 12 µL of nuclease free water to the library
RETURN LIBRARY TO FREEZER
Unwrap e-gel, remove comb, remove camera (top) from the Power Snap unit and load e-gel into machine
Pipette all 20 µL of diluted ladder from parafilm into lane 1 of e-gel
Pipette all 20 µL of diluted RAD library from parafilm into lane 2 of e-gel
Pipette 20 µL of water into remaining empty wells of e-gel
Close lid, turn Power Snap on, select "E-gel 0.8-2%" program, push start, put camera back on Power Snap unit
Set timer for 15 min
Note
The program's duration is 26 min but the gel only needs to be run for 10-15min
When timer goes off, tap "view gel" -> adjust brightness to max ->Take picture -> Tap "Export"-> insert thumb drive into usb port -> Tap "Export"
Paste e-gel picture in the BestRAD_library_prep_labData v1.1 spreadsheet under “Final Library” tab
Crop picture to include only lane1 and lane2
Use this image to determine approximate size of library and enter it into “Final Library” tab
size of library should be ~300-500bp
Expected result
21 µL of RAD library at >= 4 ng/µl and a size ~ 300- 500bp
qPCR library quantification
Note
Equipment needed
p10 pipette and tips
p200 pipette and tips
repeater M-4 pipette
1.5mL Lo-Bind microcentrifuge tubes (Fisher Scientific, 13-698-791)
Applied Biosystems MicroAmp Optical 7500 Plate (Fisher Scientific 43-168-13)
MicroAmp Optical Adhesive Film (Fisher Scientific 43-119-71)
vortex
benchtop centrifuge
0.1 mL Eppendorf combi-tips (Fisher Scientific; 13-683-700)
KimWipes
thumb drive
Equipment
7500 Real Time PCR system
NAME
qPCR
TYPE
Thermo Scientific
BRAND
discontinued
SKU
Reagents needed
NEBNext Library Quant Kit for Illumina - 500 rxnsNew England BiolabsCatalog #E7630L
Invitrogen Nuclease-Free waterFisher ScientificCatalog #43-879-36
TE pH 8.0 (1X TE Solution)IDT TechnologiesCatalog #11-01-02-05
Thaw reagents in NEBNext library quantification kit and the RAD library
Gently vortex the library, quick spin
Check small bottle in fridge for 1X dilution of qPCR dilution buffer from NEBNext Library Quant kit. If low, make more:
150 µL 10X Buffer
1350 µL nuclease-free water
Make sure the master mix in the qPCR kit has been prepped. Look for the "Prep checkbox". If it hasn't, follow instructions below
Make serial dilutions in 1.5 Lo-Bind tubes as follows:
Note
The accuracy of the qPCR depends on the accuracy and precision of the preparation of the serial dilutions below. In the following steps, each time a dilution is made, it should be thoroughly mixed but you also want to avoid bubbles forming. Annoying, yes.
Note
If the Qubit score from Step 24.5 was > 25 ng/µl, use dilutions starting from 1:100,000 or 1:200,000 in qPCR
If the Qubit score was > 30 ng/µl, make addition dilutions up to 1:2,000,000 dilution
If the Qubit score was high, you may not want to run the more concentrated dilutions (1 and 2).
1. 1:1000 dilution: 1 µL RAD library +999 µL 1X Buffer
2. 1:10,000 dilution: 10 µL of 1:1000 dilution + 90 µL 1X Buffer
3. 1:100,000 dilution:10 µL of 1:10,000 dilution + 90 µL 1X Buffer
4. 1:200,000 dilution:50 µL of 1:100,000 dilution + 50 µL 1X Buffer
5. 1:500,000 dilution: 20 µL of 1:100,000 dilution +80 µL 1X Buffer
6. 1:1,000,000 dilution: 10 µL of 1:100,000 dilution + 90 µL 1X Buffer
7. 1:2,000,000 dilution: 50 µL of 1:1,000,000 dilution + 50 µL 1X Buffer
Using a 0.1mL combitip, dispense 8 µL of Quant Master Mix to each well of a MicroAmp Optical 7500 Plate according to plate layout
Example of plate layout - all standards and library dilutions are in triplicate
Using a p10 pipette, add 2 µL of standards (use standards 2-5) from qPCR kit, library dilutions and no template control (NTC; 1X Buffer) to each well according to plate layout. Discard tip after each use.
Note
Add to side of well. To avoid bubbles, DO NOT pump!
Use MicroAmp Optical Adhesive Film to seal plate (this isn't heat sealed). Use a KimWipe to press down on the seal to avoid smudges
Quick spin DO NOT VORTEX
Check underside of plate for dirt/lint, check wells for bubbles
Turn on 7500 qPCR system. Push to eject tray, load plate, push to close
- Open 7500 v2.3 software
- User name: GUEST
- Click "Template" -> Open “E7630 slow_v2” (screen will be on “Plate setup”)
- In left “Setup” column: Click on “Experiment Properties”
- Enter in Experiment name (e.g. CarpRADseq_library1_09292022)
- Leave everything else as is
- In left “Setup” column: Click on “Plate Setup”
- Click on “Assign Targets and Samples” tab
- Highlight all wells that you’re not using à Right click and select “Clear”
Click on “Save as” and save to project folder on computer
Start Run (~01:00:00 )
1h
Export data.xls onto flash drive, and enter in results on NEB website to get the undiluted concentration of your RAD library
Example of Nebiocalculator – Efficiency needs to be as close to 100% as possible (+/- 10%), and R2 = 1
Note
Fragment size is the approximate band size obtained from E-gel
Dilute RAD library to 4nM using V1 = (C2*V2/V1) using 1X TE
Note
C1 = concentration of library according to qPCR
C2 = 4nM
V2 = 50 µL (This may change if you have a very high or low concentrated library. 50µl is appropriate if your library was ~150-250nM)
Keep original and diluted libraries at -20 °C
Expected result
Refer to "BESTRAD on NextSeq Protocol" for how to load the library
Citations
Omar A. Ali, Sean M. O’Rourke, Stephen J. Amish, Mariah H. Meek, Gordon Luikart, Carson Jeffres and Michael R. Miller. RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping
https://doi.org/10.1534/genetics.115.183665