Dec 09, 2019

Public workspaceBestRAD protocol

DOI
dx.doi.org/10.17504/protocols.io.6awhafe
  • 1University of Montana
  • Mimulus
Thom Nelson
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DOI: dx.doi.org/10.17504/protocols.io.6awhafe
Protocol Citation: Thom Nelson 2019. BestRAD protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.6awhafe
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: August 09, 2019
Last Modified: December 09, 2019
Protocol Integer ID: 26678
Keywords: Massively parallel sequencing, MPS, restriction-site associated DNA, RAD, sequence capture, genotyping, population genetics
Abstract
Modified from protocol of Sean O’Rourke and Mike Miller published in:
CITATION
Omar A. Ali, Sean M. O’Rourke, Stephen J. Amish, Mariah H. Meek, Gordon Luikart, Carson Jeffres and Michael R. Miller (2016). RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping. GENETICS.
LINK
https://doi.org/10.1534/genetics.115.183665

Materials
MATERIALS
ReagentCutSmart Buffer - 5.0 mlNew England BiolabsCatalog #B7204S
ReagentNEBNext Multiplex Oligos for Illumina (Index Primers Set 1) - 24 rxnsNew England BiolabsCatalog #E7335S
ReagentPstI - 10,000 unitsNew England BiolabsCatalog #R0140S
ReagentBfaI - 500 unitsNew England BiolabsCatalog #R0568S
ReagentNEBNext 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
Integrated DNA technologies
  • 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:


Safety warnings
Please refer to the SDS (Safety Data Sheet) for safety warings and hazard information.
Part 1 - Digestion and BestRAD adaptor ligation
Part 1 - Digestion and BestRAD adaptor ligation
I. Restriction Digest

Dilute genomic DNA samples to a common concentration.
  1. For multiplexes of ≥48, 5 - 10 ng/ul will suffice.
  2. Add Amount10 µL (50-100 ng) of genomic DNA to each well of a 96-well PCR plate.
Please select between the two following options:

  • performing sheared, single-digest RAD
  • performing double-digest
Step case

IF performing sheared, single-digest RAD
155 steps

Steps for performing sheared, single-digest RAD.
Into 1.5 ml tube, make restriction digest master mix (vols for 100 rxns).

  • 76.2 μL water
  • 134.4 μL 10X NEBuffer 3.1
  • 13.4 μL PstI (NEB R0140L)
Add Amount76.2 µL water .

Add Amount134.4 µL 10X NEBuffer 3.1 .

Add Amount13.4 µL PstI (NEB R0140L) .

Pipette equal volumes of master mix into each tube of an 8-tube strip.
Note
~Amount27 µL ea for full 96-well plate.


To each 96 plate well, pipet Amount2 µL PstI digestion master mix .

In a thermal cycler:

a) Temperature37 °C for Duration01:00:00 .
b) Temperature80 °C for Duration00:20:00 .
c) Slow ramp to Temperature4 °C — Temperature0.1 °C /sec
II. Adaptor Ligation
Note
This part assumes Amount20 µL ligation volume and and use of NEB3.1 in the previous step. If only CutSmart buffer was used for digestion, supplement ligation buffer with fullstrength NEB2.1 (Amount2 µL per well).

Add Amount2 µL annealed BestRad SbfI/PstI adaptors (50 nM) .
Make ligation master mix (vols for 100 rxns):

  • Amount347.2 µL water
  • Amount56 µL NEB2.1
  • Amount22.4 µL rATP (100 mM, Fermentas R0441)
  • Amount22.4 µL Ligase (NEB M0202L)
Note
From S. Bassham: NEB Buffer 2 is used in the ligation reactions in this protocol instead of ligase buffer because the salt it contains (50 mM NaCl) ensures the double-stranded adapters remain annealed during the reactions. T4 DNA Ligase is active in all 4 NEB Buffers if supplemented with 1mM rATP, but doesn’t work at maximum efficiency in NEB 3.

Add Amount347.2 µL water .
Add Amount56 µL NEB2.1 .
Add Amount22.4 µL rATP (100 mM, Fermentas R0441) .
Add Amount22.4 µL Ligase (NEB M0202L) .
Pipette Amount56 µL master mix into each tube of an 8-tube strip for multichannel distribution.

Into each 96 plate well, pipet Amount4 µL ligation master mix .
Incubate plate at Temperature20 °C overnight.
III. NEXT DAY

Heat kill the ligation
  1. Kill the ligation reactions by deactivating ligase at Temperature65 °C .
  2. In thermal cycler, incubate plate at Temperature65 °C for Duration00:20:00 .
Note
On Fishman Lab thermal cyclers, program available at THOM>LIGKILL.

Kill the ligation reactions by deactivating ligase at Temperature65 °C .
In thermal cycler, incubate plate at Temperature65 °C for Duration00:20:00 .
Multiplex 48 barcoded samples

1. Using an 8-channel pipettor:
a) transfer Amount10 µL of each adaptor-ligated sample into an 8-tube PCR strip.
2. Combine pooled samples into a single 1.5 mL tube.
3. Split the multiplex into two or more aliquots.
a) The following steps assume Amount200 µL multiplexed DNA .
4. Store plate at Temperature-20 °C for future multiplexing.



Using an 8-channel pipettor, transfer Amount10 µL of each adaptor-ligated sample into an 8-tube PCR strip.

Combine pooled samples into a single 1.5 mL tube.
Split the multiplex into two or more aliquots.
Note
The following steps assume Amount200 µL multiplexed DNA .

Store plate at Temperature-20 °C for future multiplexing.
Concentrate sample to 210 μL with Ampure 1X. Divide sample into two Bioruptor tubes.
Sonicate with BioRuptor NGS:
9 cycles: Duration00:00:30 on , Duration00:01:30 off


Run Amount2 µL on the fragment analyzer NGS mode to assay shearing efficiency.
Part 2 - RAD-tag isolation
Part 2 - RAD-tag isolation
I. Before beginning the following steps, set a wet or dry bath to Temperature56 °C .
II. Prepare Dynabead M280 streptavidin magnetic beads.

Adjust bead volume.
2X Binding and Wash (B+W) Buffer.

  • Concentration10 Mass Percent TrisHCl (pH 7.5)
  • Concentration1 Mass Percent EDTA pH 8.0
  • Concentration2 Mass Percent NaCl
Transfer Amount30 µL Dynabeads to a new 1.7 ml tube.

Place tube in magnetic rack and remove supernatant.
Wash the beads (1/2)

  1. Amount100 µL 2X B+W buffer
  2. Mix Duration00:00:30
  3. Quick spin
  4. Remove supernate
Add Amount100 µL 2X B+W buffer .
Mix for Duration00:00:30 .
Quick spin.
Remove supernate.
Wash the beads (2/2)

  1. Amount100 µL 2X B+W buffer
  2. Mix Duration00:00:30
  3. Quick spin
  4. Remove supernate
Add Amount100 µL 2X B+W buffer .
Mix for Duration00:00:30 .
Quick spin.
Remove supernate.
Resuspend beads in X μL 2X B+W buffer (X = multiplex volume).
III. Bead Binding

Add resuspended beads to ~Amount200 µL multiplexed DNA .

While DNA fragments are binding, make a dilution BW buffer 2-fold in 10 mM Tris.

1. Will need ~ Amount1 mL per multiplex
2. Split dilution into 2x 1.5mL tubes and set one tube in a Temperature56 °C wet or dry bath.
Incubate at TemperatureRoom temperature for Duration00:20:00 .

Mix every Duration00:02:00 by inverting.
Quick spin

  1. Remove liquid from cap of tube
  2. Not enough to pellet beads
Place tube on magnetic rack - Wash (1/4)

  1. Remove supernate.
  2. Resuspend beads in Amount150 µL 1X B+W Buffer .
Place tube on magnetic rack - Wash (2/4)

  1. Remove supernate.
  2. Resuspend beads in Amount150 µL 1X B+W Buffer .
Place tube on magnetic rack - Wash (3/4)

  1. Remove supernate.
  2. Resuspend beads in Temperature56 °C Amount150 µL 1X B+W Buffer .

Place tube on magnetic rack - Wash (4/4)
  1. Remove supernate.
  2. Resuspend beads in Temperature56 °C Amount150 µL 1X B+W Buffer .
IV. Liberate DNA from beads

Dilute an aliquot of appropriate NEBuffer 10-fold to 1X.
Resuspend beads in Amount100 µL 1X restriction digest buffer (NEBuffer 3.1 for PstI).
Place on magnetic rack, remove supernate.
Resuspend beads in Amount40 µL 1X digest buffer .

Transfer to PCR tube.
Add Amount2 µL PstI .
Incubate tube at Temperature37 °C for Duration01:00:00 .
Note
On Fishman Lab thermal cyclers, program available at THOM>CUTSMART.

Note
Because the next step removes the enzyme, there is no need to heat inactivate.

Quick spin.
Place tube on magnetic rack, KEEP SUPERNATE!!
V. Bead cleanup of digestion reaction

Allow an aliquot of Ampure XP beads to warm to TemperatureRoom temperature for ~Duration00:30:00 .
Vortex prior to addition to resuspend.
Add Amount40 µL Ampure XP beads to ligation reaction.

1. Mix very well by pipetting
a) slowly
b) expel final volume slowly due to viscosity
2. Allow to incubate at TemperatureRoom temperature for Duration00:10:00 .
Add Amount40 µL Ampure XP beads to ligation reaction.
Mix very well by pipetting.
  • slowly
  • expel final volume slowly due to viscosity
Allow to incubate at TemperatureRoom temperature for Duration00:10:00 .
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Note
Use aliquoted ethanol in 50 ml conical tubes to limit evaporation/hydration of ethanol.

Place on magnetic rack and allow beads to pellet.
Slowly remove supernate.
Ethanol wash (1/2)
  1. Add Amount200 µL 80% ethanol .
  2. Incubate for Duration00:00:30 with beads still on magnet.
Add Amount200 µL 80% ethanol .
Incubate for Duration00:00:30 with beads still on magnet.
Ethanol wash (2/2)
  1. Add Amount200 µL 80% ethanol .
  2. Incubate for Duration00:00:30 with beads still on magnet.
Add Amount200 µL 80% ethanol .
Incubate for Duration00:00:30 with beads still on magnet.
Remove all ethanol and allow beads to dry ~ Duration00:05:00 with the lid open.
Elute DNA

1. Add Amount55 µL 10 mM Tris-HCl to bead pellet.
2. Wash buffer over beads and pipette to resuspend.
a) Suspension will turn a pale, even brown.
3. Incubate at TemperatureRoom temperature for Duration00:10:00 .
4. Place tube on magnet to separate beads.
5. Pipette supernate and place in a clean PCR tube.
Add Amount55 µL 10 mM Tris-HCl to bead pellet.
Wash buffer over beads and pipette to resuspend.
Note
Suspension will turn a pale, even brown.

Incubate at TemperatureRoom temperature for Duration00:10:00 .
Place tube on magnet to separate beads.
Pipette supernate and place in a clean PCR tube.
VI. Now is an okay time to stop if need be. Store eluted DNA at Temperature-20 °C overnight.

Pause
Part 3 - NextGen library prep
Part 3 - NextGen library prep

Note
Use full reactions NEBNext Ultra DNA Library Prep Kit for Illumina (NEB E7370S/L) with no modifications except
A. Use 1:10 diluted adaptor.
B. Especially if performing double-digest, use a molecular barcoded i5 adaptor (see Materials)

I. End-repair and A-tailing
Note
This step blunts single-stranded DNA ends and adds a single A overhang.

To the liberated DNA, add:
1. Amount3 µL NEBNext Ultra II End Prep Enzyme Mix
2. Amount7 µL NEBNext Ultra II End Prep Reaction Buffer
Add Amount3 µL NEBNext Ultra II End Prep Enzyme Mix to the liberated DNA.
Add Amount7 µL NEBNext Ultra II End Prep Reaction Buffer to the liberated DNA.
. Mix by pipetting 10x w/ a pipette set to 50 µl.
In a thermal cycler:

  • Duration00:30:00 at Temperature20 °C
  • Duration00:30:00 at Temperature65 °C
  • Hold at Temperature4 °C
Note
On Fishman Lab thermal cyclers, use THOM>NEB_EP.

II. Adaptor ligation
Note
1. This part adds universal Illumina adaptors onto all end-prepped fragments. NEB NEXT adaptors form hairpins and contain a single uracil in the loop. The USER enzyme mix has endonuclease activity that cleaves the adaptor at the U, creating a Y-shaped adaptor for subsequent addition of oligos and amplification by PCR.

2. From NEB: “If input DNA ≤ 100 ng, use 1:10 diluted adaptor (diluted in 10 mM Tris, 10 mM NaCl)”. Efficient RAD preps will isolate ~1%-15% of genomic DNA. Good luck getting anything greater than 100 ng total from 48 samples!

Make sure Ligation Master Mix is well-mixed prior to addition.
  • Vortex quickly, follow with quick spin.
Vortex quickly.
Quick spin.
To end-prepped sample, add:

1. Amount30 µL NEBNext Ultra II Ligation Master Mix
2. Amount1 µL NEBNext Ligation Enhancer
3. Amount2.5 µL NEBNext Adaptor for Illumina (diluted 1:10 from stock)
Add Amount30 µL NEBNext Ultra II Ligation Master Mix .
Add Amount1 µL NEBNext Ligation Enhancer .
Add Amount2.5 µL NEBNext Adaptor for Illumina (diluted 1:10 from stock) .

Mix sample by pipetting 10X with a pipette set to 80 µl.
Quick spin to collect any liquid from the side of the tube.
In a thermal cycler:

1. Either
a) keep the thermal cycler lid open OR
b) manually turn off heated lid
2. Duration00:15:00 at Temperature20 °C
Note
On Fishman Lab thermal cyclers, use THOM>NEB_LIG.

Add Amount3 µL USER enzyme to the ligation mixture.
Mix sample by pipetting 10X with a pipette set to 80 µl.
In a thermal cycler with heated lid:
1. Duration00:15:00 at Temperature37 °C
Note
On Fishman Lab thermal cyclers, use THOM>NEBUSER.

III. Bead cleanup of ligation reaction

Allow an aliquot of Ampure XP beads to warm to TemperatureRoom temperature for ~Duration00:30:00 .
Vortex prior to addition to resuspend.
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Add Amount87 µL Ampure XP beads to ligation reaction.
1. Mix very well by pipetting.
a) slowly
b) expel final volume slowly due to viscosity
2. Allow to incubate at TemperatureRoom temperature for Duration00:10:00 .

Add Amount87 µL Ampure XP beads to ligation reaction.
Mix very well by pipetting.
a) slowly
b) expel final volume slowly due to viscosity
Allow to incubate at TemperatureRoom temperature for Duration00:10:00 .
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Place on magnetic rack and allow beads to pellet.
Slowly remove supernate.
Ethanol wash (1/2)

  1. Add Amount200 µL 80% ethanol .
  2. Incubate for Duration00:00:30 with beads still on magnet.
Add Amount200 µL 80% ethanol .
Incubate for Duration00:00:30 with beads still on magnet.
Ethanol wash (2/2)

  1. Add Amount200 µL 80% ethanol .
  2. Incubate for Duration00:00:30 with beads still on magnet.
Add Amount200 µL 80% ethanol .
Incubate for Duration00:00:30 with beads still on magnet.
Remove all ethanol and allow beads to dry ~ Duration00:05:00 with the lid open.
Elute DNA

  1. Add Amount17 µL 10 mM Tris-HCl to bead pellet.
  2. Wash buffer over beads and pipette to resuspend.
  3. Incubate at TemperatureRoom temperature for Duration00:10:00 .
  4. Place tube on magnet to separate beads.
  5. Pipette supernate and place in a a clean PCR tube.
Add Amount17 µL 10 mM Tris-HCl to bead pellet.
Wash buffer over beads and pipette to resuspend.
Incubate at TemperatureRoom temperature for Duration00:10:00 .
Place tube on magnet to separate beads.
Pipette supernate and place in a a clean PCR tube.
IV. PCR enrichment of adaptor-ligated fragments

To the purified DNA, add:

  1. Amount25 µL NEBNext Ultra II Q5 Master Mix
  2. Amount5 µL i7 index primer
  3. Amount5 µL i5 universal primer — USE i5 WITH MOLECULAR BARCODE
Add Amount25 µL NEBNext Ultra II Q5 Master Mix to the purified DNA.

Add Amount5 µL i7 index primer to the purified DNA.
Add Amount5 µL i5 universal primer to the purified DNA. — USE i5 WITH MOLECULAR BARCODE
Mix by pipetting 40 µl 10X.
Split reaction into 2 x 25 µl reactions and run separately to reduce PCR bias.
In a thermal cycler:

1. Temperature98 °C for Duration00:00:30 — Denaturation Cycle
2. Amplification Cycles — 8-12 cycles depending on input
a) Temperature98 °C for Duration00:00:10
b) Temperature65 °C for Duration00:01:15
3. Temperature65 °C for Duration00:05:00 — Final extension cycle
4. Temperature4 °C forever
V. Bead cleanup of PCR

Allow an aliquot of Ampure XP beads to warm to TemperatureRoom temperature for ~Duration00:30:00 .
Vortex prior to addition to resuspend.
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Add Amount45 µL Ampure XP beads to ligation reaction.

1. Mix very well by pipetting.
a) slowly
b) expel final volume slowly due to viscosity
2. Allow to incubate at TemperatureRoom temperature for Duration00:10:00 .
Add Amount45 µL Ampure XP beads to ligation reaction.
Mix very well by pipetting.
a) slowly
b) expel final volume slowly due to viscosity
Allow to incubate at TemperatureRoom temperature for Duration00:10:00 .
Make fresh 80% ethanol. ≥ 1 mL per reaction.
Place on magnetic rack and allow beads to pellet.
Slowly remove supernate.
Ethanol wash (1/2)

  1. Add Amount200 µL 80% ethanol .
  2. Incubate for Duration00:00:30 with beads still on magnet.
Add Amount200 µL 80% ethanol .
Incubate for Duration00:00:30 with beads still on magnet.
Ethanol wash (2/2)

  1. Add Amount200 µL 80% ethanol .
  2. Incubate for Duration00:00:30 with beads still on magnet.
Add Amount200 µL 80% ethanol .
Incubate for Duration00:00:30 with beads still on magnet.
Remove all ethanol and allow beads to dry ~ Duration00:05:00 with the lid open.

Elute DNA

  1. Add Amount33 µL 10 mM Tris-HCl to bead pellet.
  2. Wash buffer over beads and pipette to resuspend.
  3. Incubate at TemperatureRoom temperature for Duration00:10:00 .
  4. Place tube on magnet to separate beads.
  5. Pipette supernate and place in a a clean PCR tube.
Add Amount33 µL 10 mM Tris-HCl to bead pellet.
Wash buffer over beads and pipette to resuspend.
Incubate at TemperatureRoom temperature for Duration00:10:00 .
Place tube on magnet to separate beads.
Pipette supernate and place in a a clean PCR tube.
VI. (Optional) Size selection of library

A. Inefficient digestion or shearing of genomic DNA can result in large fragments (≥ 1000 bp) making it through library prep.

B. In addition, size selection of double-digest RAD libraries can allow for customization of genomic coverage because all fragments originating from a specific RAD locus should be equal in length across samples but different from other RAD loci.

C. Use agarose gel or Blue Pippin to size select libraries. At the very least clipping out any fragments greater than 1000 bp.
Optional
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