version: September 30, 2022

A B O U T   T H I S   P R O T O C O L

This is based on the original 2bRAD method from Wang et. al 2012 for high-throughput, genome-wide SNP genotyping. Further modifications have been made by Misha Matz to simplify library prep including no intermediate purification. The updated protocol now includes a triple barcode scheme (two standard Illumina indices and one in-line ligated index) for pooling libraries in 12-plexes midway through library prep to further minimize prep costs.

The following is the present version that we use for 2bRAD library prep (thanks to Alexis Sturm). We have found that with some organisms (e.g. Montastraea cavernosa corals) even with apparently clean, high quality DNA sometimes issues arise with digestion. Due to this, we like to add an optional qPCR step where we are able to verify successful digestion/ligation reactions.

In addition to all the samples you want to sequence, 3–5 samples should be prepped in triplicate. Triplicates should be independently prepped DNA samples from the same individual. They are useful at the analysis step to assess the overall accuracy of genotyping, set quality filtering criteria, and quantify genetic relatedness/clonality between samples.

The following protocol is written for a 96-well plate. If you have more than a plate of samples, you can sequence multiple plates on the same run by using different TruSeq indices. I typically aim for 4 million raw reads/sample (so ~3.5 plates per NovaSeq S1 SR100 flow cell max) and sequence with 20% phiX spike-in.

The protocol has been split up across 3 days for ease, but it can be done over 2 days, especially if omitting qPCR/test PCR.


S U P P L I E S

Item Manufacturer Cat. #
PCR tubes, attached flat cap, 0.2 mL USA Scientific, Inc.  1402-8100
PCR 8-tube strips, attached caps, 0.2 mL USA Scientific, Inc.  1402-3900
TempPlate Semi-Skirted 96-Well PCR Plate, 0.2 mL USA Scientific, Inc.  1402-9700
BcgI - 250 units New England Biolabs, Inc.  R0545S
T4 DNA Ligase - 100,000 units New England Biolabs, Inc.  M0202L
Thermo Scientific Maxima SYBR Green qPCR Master Mix (2X), with separate ROX vial Thermo Fisher Scientific, Inc.  FERK0252
Eppendorf twin.tec Real-Time 96-Well PCR Plates Eppendorf 951022043
Eppendorf Masterclear Real-time PCR Film Eppendorf 30132947
Titanium Taq DNA Polymerase 500 Rxns TaKarRa Bio USA, Inc.  639209
dNTP Mixture 2.5 mM each TaKarRa Bio USA, Inc.  4030
Zymo DNA Clean & Concentrator-5 (Capped) Zymo Research D4014
Invitrogen Qubit Assay Tubes Thermo Fisher Scientific, Inc.  Q32856
Invitrogen Qubit 1X dsDNA BR Assay Thermo Fisher Scientific, Inc.  Q33266


D A Y   1

D I G E S T I O N

At this step genomic DNA is digested with a type IIB restriction endonuclease, producing short restriction fragments of uniform length.

We use BcgI which produces 36 bp fragments with 2 bp overhangs.



  1. Prepare dilutions of samples at 25 ng/µL >4 µL total volume

  2. Prepare BcgI digestion master mix

    • NEB BcgI now comes with SAM in the buffer, so we replaced the SAM volume in the reaction with NFW

  3. Add 2 µL of mastermix to each well, can be accomplished quickly and precisely with a 10 µL electronic pipette

  4. Use a multi-channel pipette to combine 4 µL of the DNA sample with the 2 µL master mix (6 µL total volume)

  5. Cover the plate with PCR film, spin down, and incubate at 37 °C in a thermocycler with heated lid for 1 hr

  6. Inactivate the enzyme at 65 °C for 10 min then hold samples at 4 °C

    • Hold samples on ice from here on out

Digestion master mix recipe
Component 1 rxn (µL) 96 rxn (µL)
NFW 0.8 84.48
NEB Buffer #3 0.6 63.36
BcgI (2 U/µL) 0.6 63.36
Total: 2.0 211.20
Digestion profile
37 ºC 60 min
65 ºC 10 min
4 ºC ∞




L I G A T I O N

In this step adaptors are ligated with T4 DNA ligase to the restriction fragments produced from the above digestion.

We will use 12 uniqely indexed adaptors on the 3’ end, which will allow us to pre-pool for amplification.



  1. Prepare double stranded adaptors by combining each pair of primers, Adaptor 1 (5ILL-NNRW, anti-5ill-NNRW) and the 12 different pairs of Adaptor 2 (3ill-BC, anti-3ill-BC))

    • Pre-mixed adaptors can be stored at -20 ºC for several months

Adaptor 1
Component 1 rxn (µL) 96 rxn (µL)
5ill-NNRW (10 µM) 0.5 60
anti-5ill-NNRW (10 µM) 0.5 60
Total: 1.0 120
Adaptor 2 (1 per plate column)
Component 1 rxn (µL) 8 rxn (µL)
3ill-BC(1–1; 10 µM) 0.5 5
anti-3ill-BC(1–12; 10 µM) 0.5 5
Total: 1.0 10


  1. Incubate at 42 °C for 5 minutes then keep at room temperature until ligation

  2. Prepare 12 master mixes for ligations (one for each indexed 3’ adaptor)

Ligation recipe
Component 1 rxn (µL)
NFW 15
10X T4 ligase buffer w/ 10 mM ATP 2
Adaptor 1 (5 μM) 1
Adaptor 2 (5 μM; 1 per plate column) 1
T4 DNA ligase 1
Total: 20
Ligation profile
16 ºC 12 hr
65 ºC 60 min
4 ºC ∞







  • It’s easiest to first make an initial master mix:
Ligation master mix recipe
Component 1 rxn (µL) 8 rxn (µL) 12 MM (µL)
10X T4 DNA ligase buffer w/ 10 mM ATP 2 17.6 232.3
Adaptor 1 (5 μM) 1 8.8 116.2
T4 DNA ligase 1 8.8 116.2
Total: 4 35.2 464.6


  • Then, into 12 separate 0.6 mL tubes (or PCR tubes) add the above Ligation Master Mix with one of the 12 indexed 3ill-BC adaptors, yilelding 12 uniquely indexed ligation reactions:
3ill-BC(1–12) indexed ligation recipe
Component 8 rxn (µL)
NFW 132.0
Ligation Master Mix 35.2
5 μM Adaptor 2 (1 per plate column) 8.8
Total: 176.0


  1. Add 20 µL master mix to digested DNA ( now 26 µL total volume)

    • Each uniquely indexed ligation is added down a column on the 96-well plate

    • Can use multi-chanel pipette to quickly combine

    • Keep on ice while mixing


  1. Incubate at 16 °C for 12 hours

  2. Heat at 65 °C for ≥ 40 min to inactivate the ligase (in a thermocycler w/ heated lid)


D A Y   2

O P T I O N A L   q P C R

We use qPCR to verify successful digestion/ligation.
You can amplify samples in duplicate to get an average CT, but I like to use an electronic multi-chanel pipette and only use 1 well per sample on a qPCR plate.

qPCR master mix recipe
Component 1 rxn (µL)
NFW 5.53
SYBR Green Mastermix 7.50
TruSeq oligo (10 µM) 0.07
Any ILL-BC oligo (1 µM ) 0.70
P5 (10 µM) 0.10
P7 (10 µM) 0.10
Total: 14.00
qPCR profile
95 ºC 10 min
95 ºC 15 s
60 ºC 30 s 40 cycles
72 ºC 30 s







  1. Add 14 µL of master mix to each well, avoiding bubbles

    • If you are accurate with pipeting, you can split a full plate into 2 qPCR plates with 2–3 no-template control wells per qPCR plate

    • Otherwise you can run three rows of samples in duplicate and 2–3 no-template control wells on each qPCR plate

  2. Add 1 µL of ligation to each well, spin down plate

  3. Turn on qPCR machine, ensure the correct reaction volume (15 µL) is input

  4. Rank samples from highest to lowest CT score.

  5. Optional: Select 4 sample ligations with relatively low CT scores and 4 samples with relatively high CT scores to use in a test PCR.


O P T I O N A L   T E S T   P C R

A test PCR can be run to verify successful ligation, or to see what CT values are too low before pooling ligations.

For a set of 8 ligations, we recommended using 4 with low CT values and 4 with high CT values if qPCR was conducted.

I like to do atleast 1 test PCR for every new organism sequenced to ballpark good/bad CT values.

Test PCR master mix
Component 1 rxn (µL) 8 rxn (µL)
NFW 9.7 85.36
10 mM dNTP mix 0.4 3.52
P5 (10 µm) 0.4 3.52
P7 (10 µM) 0.4 3.52
ILL-BC oligo (1 µM) 2.4 21.12
TruSeq oligo (10 µM) 0.3 2.64
10X Titanium buffer 2.0 17.60
Titanium Taq 0.4 3.52
Total: 16.0 140.80
PCR profile
70 °C 30 s
95 °C 20 s
65 °C 3 min 15 Cycles
72 °C 30 s
4 °C ∞








  1. Add 16 µL of master mix to 8 strip-tubes, then add 4 µL of ligation and amplify

  2. Load 5 µL on a 2% agarose gel alongside LMW ladder or other marker that has 150 and 200 bp bands, confirm that all samples have a visible band at ~180 bp


D A Y   3

A M P L I F I C A T I O N

In this step, the constructs produced by ligation are amplified using a set of four primers that introduce pooled, sample-specific barcodes and the annealing sites for HiSeq/NovaSeq amplification and sequencing primers.



  1. Pool ligations across row into strip-tubes, using 6 µl from each well

    • Using a multi-chanel pipette speeds up pooling

    • The 96-well plate is now reduced to 8 pooled samples, each corresponding to the original row

    • If needed pooled ligations can be stored at 4 ºC



  1. For each reaction prepare the following master mix:
PCR master mix recipe
Component 1 pool (µL) 8 pools (µL)
NFW 14.4 126.72
10 mM dNTP mix 1.0 8.80
P5 (10 µM) 1.0 8.80
P7(10 µM ) 1.0 8.80
TruSeq (10 µM) 0.6 5.28
10X Titanium buffer 5.0 44.00
Titanium Taq 1.0 8.80
Total: 24.0 211.20


  1. Set up 8 PCR tubes, combine and amplify:
2bRAD PCR
Component 1 pool (µL)
Master Mix 24
Pooled Ligation 20
1 µM ILL-BC primer (Different for each pool) 6
Total: 50
PCR profile
70 °C 30 s
95 °C 20 s
65 °C 3 min 15 Cycles
72 °C 30 s
4 °C ∞


  1. Load 5 µL on a 2% agarose gel alongside LMW ladder (NEB N3233S) or other marker that has 150 and 200 bp bands

    • There should be no substantial variation in product amount among samples at this stage

  2. Confirm that all samples have a visible band at ~180 bp. You might also see a band below 150 bp, which is an artifact from the carried-over ligase (if this is an issue you can heat inactivate the ligase for longer

  3. If the 180 bp product is visible but barely, add two more cycles to the same reactions

    • Do not continue to amplify the samples if no band is visible at 15 cycles, go back and troubleshoot the samples within that pool


  1. Pool 20 µL of each library and purify/concentrate for sequencing

    • I use Zymo DCC-5 with Binding Buffer:PCR product ratio of 5:1 and elute in ~25 µL NFW

  2. Quantify cleaned/concentrated pool with Qubit for sequencing


O L I G O S


Name Sequence Index Scale Purification
5ILL-NNRW0 CTACACGACGCTCTTCCGATCTNNRWNN – 25nm STD
anti-5ill-NNRW WYNNAGATCGGA/3InvdT/ – 100nm STD
3ill-BC1 CAGACGTGTGCTCTTCCGATCTACACNN ACAC 25nm STD
3ill-BC2 CAGACGTGTGCTCTTCCGATCTGTCTNN GTCT 25nm STD
3ill-BC3 CAGACGTGTGCTCTTCCGATCTTGGTNN TGGT 25nm STD
3ill-BC4 CAGACGTGTGCTCTTCCGATCTCACTNN CACT 25nm STD
3ill-BC5 CAGACGTGTGCTCTTCCGATCTGATGNN GATG 25nm STD
3ill-BC6 CAGACGTGTGCTCTTCCGATCTTCACNN TCAC 25nm STD
3ill-BC7 CAGACGTGTGCTCTTCCGATCTCTGANN CTGA 25nm STD
3ill-BC8 CAGACGTGTGCTCTTCCGATCTAAGCNN AAGC 25nm STD
3ill-BC9 CAGACGTGTGCTCTTCCGATCTGTAGNN GTAG 25nm STD
3ill-BC10 CAGACGTGTGCTCTTCCGATCTGACANN GACA 25nm STD
3ill-BC11 CAGACGTGTGCTCTTCCGATCTGTGANN GTGA 25nm STD
3ill-BC12 CAGACGTGTGCTCTTCCGATCTAGTCNN AGTC 25nm STD
anti-3ill-BC1 GTGTAGATCGGA/3InvdT/ GTGT 100nm STD
anti-3ill-BC2 AGACAGATCGGA/3InvdT/ AGAC 100nm STD
anti-3ill-BC3 ACCAAGATCGGA/3InvdT/ ACCA 100nm STD
anti-3ill-BC4 AGTGAGATCGGA/3InvdT/ AGTG 100nm STD
anti-3ill-BC5 CATCAGATCGGA/3InvdT/ CATC 100nm STD
anti-3ill-BC6 GTGAAGATCGGA/3InvdT/ GTGA 100nm STD
anti-3ill-BC7 TCAGAGATCGGA/3InvdT/ TCAG 100nm STD
anti-3ill-BC8 GCTTAGATCGGA/3InvdT/ GCTT 100nm STD
anti-3ill-BC9 CTACAGATCGGA/3InvdT/ CTAC 100nm STD
anti-3ill-BC10 TGTCAGATCGGA/3InvdT/ TGTC 100nm STD
anti-3ill-BC11 TCACAGATCGGA/3InvdT/ TCAC 100nm STD
anti-3ill-BC12 GACTAGATCGGA/3InvdT/ GACT 100nm STD
P5 AATGATACGGCGACCACCGA – 25nm STD
P7 CAAGCAGAAGACGGCATACGA – 25nm STD
TruSeq_Un1 AATGATACGGCGACCACCGAGATCTACACATCACGACACTCTTTCCCTACACGACGCTCTTCCGATCT ATCACG 20nmU STD
TruSeq_Un2 AATGATACGGCGACCACCGAGATCTACACACTTGAACACTCTTTCCCTACACGACGCTCTTCCGATCT ACTTGA 20nmU STD
TruSeq_Un3 AATGATACGGCGACCACCGAGATCTACACTAGCTTACACTCTTTCCCTACACGACGCTCTTCCGATCT TAGCTT 20nmU STD
TruSeq_Un4 AATGATACGGCGACCACCGAGATCTACACGGCTACACACTCTTTCCCTACACGACGCTCTTCCGATCT GGCTAC 20nmU STD
TruSeq_Un5 AATGATACGGCGACCACCGAGATCTACACTTAGGCACACTCTTTCCCTACACGACGCTCTTCCGATCT TTAGGC 20nmU STD
TruSeq_Un6 AATGATACGGCGACCACCGAGATCTACACCGATGTACACTCTTTCCCTACACGACGCTCTTCCGATCT CGATGT 20nmU STD
ILL-BC23 CAAGCAGAAGACGGCATACGAGATCCACTCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT CCACTC 20nmU STD
ILL-BC24 CAAGCAGAAGACGGCATACGAGATGCTACCGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT GCTACC 20nmU STD
ILL-BC25 CAAGCAGAAGACGGCATACGAGATATCAGTGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT ATCAGT 20nmU STD
ILL-BC26 CAAGCAGAAGACGGCATACGAGATGCTCATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT GCTCAT 20nmU STD
ILL-BC27 CAAGCAGAAGACGGCATACGAGATAGGAATGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT AGGAAT 20nmU STD
ILL-BC28 CAAGCAGAAGACGGCATACGAGATCTTTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT CTTTTG 20nmU STD
ILL-BC29 CAAGCAGAAGACGGCATACGAGATTAGTTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT TAGTTG 20nmU STD
ILL-BC30 CAAGCAGAAGACGGCATACGAGATCCGGTGGTGACTGGAGTTCAGACGTGTGCTCTTCCGAT CCGGTG 20nmU STD