Jul 08, 2020
Single-strand library preparation protocol
- 1W. Szafer Institute of Botany, Polish Academy of Sciences
Protocol Citation: Tomasz Suchan 2020. Single-strand library preparation protocol. protocols.io https://dx.doi.org/10.17504/protocols.io.q5ydy7w
Manuscript citation:
Tin MM-Y, Economo EP, Mikheyev AS (2014) Sequencing Degraded DNA from Non-Destructively Sampled Museum Specimens for RAD-Tagging and Low-Coverage Shotgun Phylogenetics. PLoS ONE 9(5): e96793. https://doi.org/10.1371/journal.pone.0096793
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: June 20, 2018
Last Modified: July 08, 2020
Protocol Integer ID: 13208
Guidelines
This library preparation protocol can be easily adapted for different indexing strategies. Combinations of indexed primes can be used for generating single- or double-indexed libraries. In the first case, PCR_F_universal primer is used in combination with PCR_R_**** primers. For double indexed libraries, the combinations of PCR_F_**** and PCR_R_**** can be used for up to 384 combinations with the primers listed below.
Additionally, libraries can be prepared by adding inline index (barcode) to the adapters and amplified either with unindexed illumina primers, or single- or double-indexing strategy. This sequence is then present at the begining of the reads and can be used for sample demultiplexing.
Finally, by combining inline barcode with indexed forward and reverse primers, triple-indexed libraries can be constructed. This design is especially useful for the libraries used for the sequence-capture protocols. In such experiments, the reads are captured on DNA or RNA baits and reamplified afterwards. If performed on pooled samples, the post-capture PCR can cause chimeric reads formation among hopmologous sequences from different specimans. Triple-indexing strategy allows for controlling these by comparing the index combination used with the inline barcode.
Adapter sequences:
Unindexed adapters:
Tin_P1_lower: [PHO]AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
Tin_P1_upper: CCCTACACGACGCTCTTCCGATCT
Inline indexed (barcoded) adapters:
Tin_P1_lower_barcoded: [PHO]nnnnnnnnAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
Tin_P1_upper_barcoded: CCCTACACGACGCTCTTCCGATCTnnnnnnnn
The barcode sequences (nnnnnnnn) can be designed using published scripts (https://bioinf.eva.mpg.de/multiplex/). To prepare 25 μM working solution, mix 50 μl of the upper and lower oligos (100 μM stock) with 40 μl of water and 10 μl of the 10x annealing buffer to obtain 100 μL of working solution. Adapters have to be annealed as follows: heat to 95°C for 1 minute and slowly bring to 20°C with a ramp of 0.1°C/s.
Oligo used in the second-strand synthesis step:
Tin_P2-C5: GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTCCCCC
Primer sequences used:
forward: AATGATACGGCGACCACCGAGATCTACACnnnnnnnACACTCTTTCCCTACACGACGC
reverse: CAAGCAGAAGACGGCATACGAGATnnnnnnnGTGACTGGAGTTCAGACGTGTGC
nnnnnnn = index sequence
PCR_F_A501: AATGATACGGCGACCACCGAGATCTACACTGAACCTTACACTCTTTCCCTACACGACGC
PCR_F_A502: AATGATACGGCGACCACCGAGATCTACACTGCTAAGTACACTCTTTCCCTACACGACGC
PCR_F_A503: AATGATACGGCGACCACCGAGATCTACACTGTTCTCTACACTCTTTCCCTACACGACGC
PCR_F_A504: AATGATACGGCGACCACCGAGATCTACACTAAGACACACACTCTTTCCCTACACGACGC
PCR_F_A505: AATGATACGGCGACCACCGAGATCTACACCTAATCGAACACTCTTTCCCTACACGACGC
PCR_F_A506: AATGATACGGCGACCACCGAGATCTACACCTAGAACAACACTCTTTCCCTACACGACGC
PCR_F_A507: AATGATACGGCGACCACCGAGATCTACACTAAGTTCCACACTCTTTCCCTACACGACGC
PCR_F_A508: AATGATACGGCGACCACCGAGATCTACACTAGACCTAACACTCTTTCCCTACACGACGC
PCR_F_D501: AATGATACGGCGACCACCGAGATCTACACTATAGCCTACACTCTTTCCCTACACGACGC
PCR_F_D502: AATGATACGGCGACCACCGAGATCTACACATAGAGGCACACTCTTTCCCTACACGACGC
PCR_F_D503: AATGATACGGCGACCACCGAGATCTACACCCTATCCTACACTCTTTCCCTACACGACGC
PCR_F_D504: AATGATACGGCGACCACCGAGATCTACACGGCTCTGAACACTCTTTCCCTACACGACGC
PCR_F_D505: AATGATACGGCGACCACCGAGATCTACACAGGCGAAGACACTCTTTCCCTACACGACGC
PCR_F_D506: AATGATACGGCGACCACCGAGATCTACACTAATCTTAACACTCTTTCCCTACACGACGC
PCR_F_D507: AATGATACGGCGACCACCGAGATCTACACCAGGACGTACACTCTTTCCCTACACGACGC
PCR_F_D508: AATGATACGGCGACCACCGAGATCTACACGTACTGACACACTCTTTCCCTACACGACGC
PCR_F_universal: AATGATACGGCGACCACCGAGATCTACACTCTTTCCCTACACGACG
PCR_R_A701: CAAGCAGAAGACGGCATACGAGATGTCGTGATGTGACTGGAGTTCAGACGTGTGC
PCR_R_A702: CAAGCAGAAGACGGCATACGAGATACCACTGTGTGACTGGAGTTCAGACGTGTGC
PCR_R_A703: CAAGCAGAAGACGGCATACGAGATTGGATCTGGTGACTGGAGTTCAGACGTGTGC
PCR_R_A704: CAAGCAGAAGACGGCATACGAGATCCGTTTGTGTGACTGGAGTTCAGACGTGTGC
PCR_R_A705: CAAGCAGAAGACGGCATACGAGATTGCTGGGTGTGACTGGAGTTCAGACGTGTGC
PCR_R_A706: CAAGCAGAAGACGGCATACGAGATGAGGGGTTGTGACTGGAGTTCAGACGTGTGC
PCR_R_A707: CAAGCAGAAGACGGCATACGAGATAGGTTGGGGTGACTGGAGTTCAGACGTGTGC
PCR_R_A708: CAAGCAGAAGACGGCATACGAGATGTGTGGTGGTGACTGGAGTTCAGACGTGTGC
PCR_R_A709: CAAGCAGAAGACGGCATACGAGATTGGGTTTCGTGACTGGAGTTCAGACGTGTGC
PCR_R_A710: CAAGCAGAAGACGGCATACGAGATTGGTCACAGTGACTGGAGTTCAGACGTGTGC
PCR_R_A711: CAAGCAGAAGACGGCATACGAGATTTGACCCTGTGACTGGAGTTCAGACGTGTGC
PCR_R_A712: CAAGCAGAAGACGGCATACGAGATCCACTCCTGTGACTGGAGTTCAGACGTGTGC
PCR_R_D701: CAAGCAGAAGACGGCATACGAGATCGAGTAATGTGACTGGAGTTCAGACGTGTGC
PCR_R_D702: CAAGCAGAAGACGGCATACGAGATTCTCCGGAGTGACTGGAGTTCAGACGTGTGC
PCR_R_D703: CAAGCAGAAGACGGCATACGAGATAATGAGCGGTGACTGGAGTTCAGACGTGTGC
PCR_R_D704: CAAGCAGAAGACGGCATACGAGATGGAATCTCGTGACTGGAGTTCAGACGTGTGC
PCR_R_D705: CAAGCAGAAGACGGCATACGAGATTTCTGAATGTGACTGGAGTTCAGACGTGTGC
PCR_R_D706: CAAGCAGAAGACGGCATACGAGATACGAATTCGTGACTGGAGTTCAGACGTGTGC
PCR_R_D707: CAAGCAGAAGACGGCATACGAGATAGCTTCAGGTGACTGGAGTTCAGACGTGTGC
PCR_R_D708: CAAGCAGAAGACGGCATACGAGATGCGCATTAGTGACTGGAGTTCAGACGTGTGC
PCR_R_D709: CAAGCAGAAGACGGCATACGAGATCATAGCCGGTGACTGGAGTTCAGACGTGTGC
PCR_R_D710: CAAGCAGAAGACGGCATACGAGATTTCGCGGAGTGACTGGAGTTCAGACGTGTGC
PCR_R_D711: CAAGCAGAAGACGGCATACGAGATGCGCGAGAGTGACTGGAGTTCAGACGTGTGC
PCR_R_D712: CAAGCAGAAGACGGCATACGAGATCTATCGCTGTGACTGGAGTTCAGACGTGTGC
Demultiplexing:
For index 1 (i7) - use the reverse complementary sequence in bold from reverse primers.
For index 2 (i5) for NovaSeq, MiSeq, HiSeq 2000/2500 systems - use the sequence of the one in bold from forward primers.
For index 2 (i5) for iSeq 100, MiniSeq, NextSeq 550, NextSeq 500, HiSeq 4000, and HiSeq 3000 systems - use the reverse complementary sequence in bold from forward primers.
Before start
Primers: prepare 5 μM solutions.
Oligo used in the second-strand synthesis step (Tin_P2-C5): prepare 15 μM solution
Adapters: prepare 25 μM solutions:
- mix 50 μl of each pair of barcoded Tin_P1_lower and Tin_P1_upper (100 μM stock), 10 μl of 10x annealing buffer and 40 μl of Tris 10 mM,
- incubate both tubes in a PCR cycler: 10 s at 95°C, bring down to 12°C 0.1 C per sec; you have the annealed adapters at the concentration of 40 μM,
- adapter is now at 25 μM in 100 μl.
Make sure that you anneal the proper adapter oligos together (from the same pair)! Make sure not to crosscontaminate your barcoded adapters (use filter tips, clean bench with bleach, ideally wotk in a PCR or laminar-flow hood).
Dephosphorylation
Dephosphorylation
Prepare master mix 1:
1.3 µL NEBuffer 4 (10x)
0.7 µL water
1 µL FAST alkaline phosphatase (1 U/μl)
Add 3 μl of master mix 1 to 10 μl of DNA (total volume = 13 μl).
Incubate for 1 h at 37°C, denature the DNA for 5 min at 95°C, put immediatly on ice.
Safety information
Prepare ice at the and of incubation. Move sample immediatelly form 95°C to the ice.
Guanidine tailing
Guanidine tailing
Prepare master mix 2 (assemble at room temperature, can precipitate when on ice):
2.5 µL water
0.7 µL NEBuffer 4 (10x)
2 µL CoCl2 (2.5 mM)
0.8 µL GTP (100 mM)
1 µL TdT (20 U/μl)
Add 7 μl of the master mix 2 to the denatured DNA (total volume = 20 μl).
Incubate for 30 min at 37°C, heat-kill the enzymes for 10 min at 70°C. Spin down.
Second strand synthesis
Second strand synthesis
Prepare master mix 3:
5.4 µL water
1 µL NEBuffer 4 (10x)
0.6 µL dNTP mix (25 mM each)
1 µL P2-CCCCC oligo (15 mM)
2 µL Klenow exo- (5 U/μl)
Add 10 μl of the master mix 3 to 20 μl of the DNA (total volume = 30 μl).
Incubate at room temperature for 3 hours, heat-kill enzymes for 20 min at 75°C. Spin down.
Blunt-end reaction
Blunt-end reaction
Prepare master mix 4:
3.95 µL water
0.5 µL NEBuffer 4 (10x)
0.35 µL BSA (10 mg/ml)
0.2 µL T4 DNA polymerase (3 U/μl)
Add 5 μl of the master mix 4 to 30 μl of the DNA (total volume = 35 μl).
Safety information
Keep both DNA and the mix on the ice when assembling the reaction.
Incubate for 15 min at 12°C. Hold at 4°C and proceed directly to the next step.
Perform AMPure cleanup with the beads:sample ratio 2:1 (70 μl of the beads and 35 μl of the reaction) according to the manufacturer’s instructions. Resuspend in 11 μl of 10 mM Tris or water.
Ligation of P1 adapter
Ligation of P1 adapter
Transfer 10 μl the purified DNA to the plate/tubes.
Prepare barcoded P1 adapters (25 μM concentration).
Safety information
Each sample should be mixed with a different adapter in case the barcoded adapters are used.
Prepare master mix 5:
3.5 µL water
2.5 µL PEG-4000 (50%)
2 µL T4 ligase buffer (10x)
1 µL T4 DNA ligase (400 U/μl)
Add 1 μl of the adapter (25 μM working solution) to 10 μl of the DNA. Briefly vortex and spin.
Add 9 μl of the master mix 5 to each sample (total volume = 20 μl).
Incubate at 16°C for 3h. Spin down.
Perform AMPure cleanup with the beads:sample ratio 1:1 (20 μl of the beads and 20 μl of the reaction) according to the manufacturer’s instructions. Resuspend in 11 μl of 10 mM Tris or water.
PCR amplification
PCR amplification
Prepare master mix 4:
24.1 µL water
10 µL Q5 polymerase buffer (5x)
0.4 µL dNTP mix (25 mM each)
0.5 µL Q5 hot-start polymerase
Add 35 μl of the master mix 3 to each tube/well.
Add 2.5μl of each primer (5 μM working solutions).
Add 10 μl of the template.
Run PCR program:
30 s at 98°C
15 cycles of:
- 10 s at 98°C
- 20 s at 60°C
- 25 s at 72°C
5 min at 72°C
hold at 4°C
Note
Determine experimentally the required number of PCR cycles.
Expected result
Check reaction on a gel or Tapestation/Fragment Analyzer. Quantify using Qubit fluorimeter.