TITLE: hyRAD RNA probes preparation and capture
AUTHORS: Tomasz  Suchan, Ludovic Orlando

[DESCRIPTION]
Supplemental Information for:

Suchan, T., Kusliy, M.A., Khan, N., Chauvey, L., Tonasso-Calvière, L., Schiavinato, S., Southon, J., Keller, M., Kitagawa, K., Krause, J., Bessudnov, A.N., Bessudnov, A.A., Graphodatsky, A.S., Lamas, S.V., Wilczyński, J., Pospuła, S., Tunia, K., Nowak, M., Moskal-delHoyo, M., Tishkin, A.A., Pryor, A.J.E., Outram, A.K., Orlando, L. (2021) Performance and automation of ancient DNA capture with RNA hyRAD probes. Molecular Ecology Resources, doi: 10.1111/1755-0998.13518

[BEFORE_START]
RAD P1 adapters, SbfI/PstI/NsiI-compatible (RAD-T7-P1)
 
RAD-P1.1_T7-PstI: ACACTCTTTCCCTACACGACGCTCTTCCGATCTAATACGACTCACTATAGTGCA
RAD-P1.2_T7-PstI: [5phos]CTATAGTGAGTCGTATTAGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
 
The above oligonucleotides are annealed to form adapter compatible with SbfI, PstI or NsiI restriction enzyme overhang (3'TGCA). The restiction site is not active anymore after adapter ligation.
 
When sequencing, the first 18 nt will be the T7 promoter sequence and the next 6 nt of the cut-site. Please keep in mind that the beginning of the reads will have zero diversity (the T7 promoter sequence + cut site). Pool it with other libraries of high diversity, spike-in with a lot of PhiX or consider using several adapters with different length of N-padding before the T7 sequence (not tested yet but should work):
RAD_P1.1_pad-T7-PstI: ACACTCTTTCCCTACACGACGCTCTTCCGATC[pad]TAATACGACTCACTATAGTGCA
RAD_P1.2_pad-T7-PstI: [5phos]CTATAGTGAGTCGTATTA[pad]GATCGGAAGAGCGTCGTGTAGGGAAAGAGTGT
 
RAD P2 adapter, MseI-compatible (RAD-P2)
 
RAD_P2.1_MseI-bio: [5biotin]GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCT
RAD_P2.2_MseI: [5phos]TAAGATCGGAAGAGCGAGAACAA
 
The above oligonucleotides are annealed to form adapter compatible with MseI restriction enzyme overhang (5'TA). Restriction site is present after ligation, so the adapter can be removed by MseI before probes transcription. One of the strands is 5'biotinylated, this allows separating strands with P2 adapter ligated using streptavidine beads and discarding fragments with P1 adapters on both ends. 

PCR primers

PCR_F: AATGATACGGCGACCACCGAGAT
PCR_F_indexed: AATGATACGGCGACCACCGAGATCTACACxxxxxxxxACACTCTTTCCCTACACGACGC
PCR_R_indexed: CAAGCAGAAGACGGCATACGAGATxxxxxxxxGTGACTGGAGTTCAGACGTGTGC
PCR_R_post: CAAGCAGAAGACGGCATACGAGAT
 
Depending on the decision to use single or double indexing for your libraries and the probes use the combination of either unindexed forward and indexed reverse or both indexed primers. Use different indexed PCR primer for each sample. These indexed primers should be also different than the ones used for the preparation of genomic libraries to be captured.
 
For the post-capture PCR use combination of PCR_Fvd and PCR_Rev_post primers.
 
From 100 μM stock, prepare aliquots of each primer at 5 μM.
 
Blocking oligonucleotides

The sequences of DNA templates for blocking RNA transcription are listed below for single- and double-indexed libraries. You might need to change the poly-N fragment corresponding to the indices to match the lenth of the index you use.

For double-indexed libraries:
 
T7.prom: AGTACTAATACGACTCACTATAGG
BO.P5-multiplex: AATGATACGGCGACCACCGAGATCTACACNNNNNNNNACACTCTTTCCCTACACGACGCTCTTCCGATCTCCTATAGTGAGTCGTATTAGTACT
BO.P7-multiplex: AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTGCCTATAGTGAGTCGTATTAGTACT
 
For single-indexed libraries:
T7.prom: AGTACTAATACGACTCACTATAGG
BO.P5 AGATCGGAAGAGCGTCGTGTAGGGAAAGAGTGTAGATCTCGGTGGTCGCCGTATCATTCCTATAGTGAGTCGTATTAGTACT
BO.P7-multiplex: AGATCGGAAGAGCACACGTCTGAACTCCAGTCACNNNNNNNNATCTCGTATGCCGTCTTCTGCTTGCCTATAGTGAGTCGTATTAGTACT

Prepare 200 μM stock. These oligonucleotides are transcribed into RNA blocking oligonucleotides that target one strand of the final libraries as explained in the protocol.
 
Other solutions to prepare:
 
- 10 mM Tris-HCl pH 7.5 or PCR-grade water
- EDTA 500 mM
- SDS 10%
- TEN buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 1M NaCl)
- Wash buffer 1 (1x SSC / 0.1% SDS)
- Wash buffer 2 (0.1x SSC / 0.1% SDS)
- 2x Bind and Wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl)
 
Use RNase-free water for all the solutions!

[GUIDELINES]
You can change the number of samples used for the probes preparation by scaling the reaction.

For the capture, adjust the volumes accordingly to the numbers of libraries captured.

The final molarities in the capture reaction will be around a few pmol of probes and libraries, 50 pmol of each blocking oligo (around 10x the molarity of the DNA library), 2.3 ng of Cot-1 and 2.3 ng of salmon sperm DNA, in approximately 5.4x SSPE (0.8 M NaCl), 0.013 M EDTA, 1.05% SDS, 0.75x Denhardt's solution.
 
Literature:
 
Carpenter et al. (2013) Pulling the 1%: Whole-Genome Capture for the Targeted Enrichment of Ancient DNA Sequencing Libraries. The American Journal of Human Genetics 93, 852-864.
 
Mastretta-Yanes A, Arrigo N, Alvarez N, Jorgensen TH, Piñero D, Emerson BC (2015) Restriction site-associated DNA sequencing, genotyping error estimation and de novo assembly optimization for population genetic inference. Molecular Ecology Resources 15(1): 28–41. doi:10.1111/1755-0998.12291
 
MYcroarray MYbaits manual v3.01, December 2015, http://www.mycroarray.com/pdf/MYbaits-manual-v3.pdf
 
Peterson BK, Weber JN, Kay EH, Fisher HS, Hoekstra HE (2012) Double Digest RADseq: An Inexpensive Method for De Novo SNP Discovery and Genotyping in Model and Non-Model Species. PLoS ONE 7(5): e37135. doi:10.1371/journal.pone.0037135
 
Suchan T, Pitteloud C, Gerasimova NS, Kostikova A, Schmid S, Arrigo N, Pajkovic M, Ronikier M, Alvarez N (2016) Hybridization Capture Using RAD Probes (hyRAD), a New Tool for Performing Genomic Analyses on Collection Specimens. PLoS ONE 11(3): e0151651. doi:10.1371/journal.pone.0151651

[STEPS]
SECTION: Preparation
1. Choose a few fresh samples coming from different populations (if possible, to capture the diversity of the targeted species). The DNA samples should be diluted to the same concentrations, ideally at 20-50 ng/μl. If you need to sequence the probes library, you should keep the samples separated for the next steps, until they are indexed in a PCR.
SECTION: Restriction
2. Prepare master mix 1:
66 1
10 1
2 1
2 1
SECTION: Restriction
3. Mix 20 μl of DNA with 80 μl of master mix A1 (total volume = 100 μl).
SECTION: Restriction
4. Incubate 3 hours at 37°C, hold at 4°C.
SECTION: Restriction
5. Perform AMPure cleanup with the beads:sample ratio 2:1 according to the manufacturer’s instructions. Resuspend in 20 μl of 10 mM Tris.
SECTION: Adapter ligation
6. Prepare RAD-P1 adapters. You need to anneal:
RAD-P1.1_T7-PstI with RAD-P1.1_T7-Pst2 to obtain RAD-T7-P1 adapter (10 μM) 
RAD_P2.1_MseI-bio with RAD_P2.2_MseI to obtain RAD-P2 adapter (10 μM)

 
Heat the mix for 1 min at 95°C, cool down to 10°C with ramp 0.1°C/s.

The annealed adapters can be stored long-term at -20°C.
SECTION: Adapter ligation
7. Prepare master mix 2:
0.2 1
2.8 1
2.0 1


SECTION: Adapter ligation
8. Mix 19 μl of digested DNA with 2 μl of RAD-T7-P1 adapter (10 μM) and 2 μl of RAD-P2 adapter (10 μM). Briefly vortex and spin.
SECTION: Adapter ligation
9. Add 5 μl of master mix 2 to the digested DNA with adapters (total volume = 28 μl). Briefly vortex and spin.
SECTION: Adapter ligation
10. Incubate 3 hours at 16°C, hold at 4°C.
 
SECTION: Adapter ligation
11. Perform AMPure cleanup with the beads:sample ratio 1.5:1 according to the manufacturer’s instructions. Resuspend in 60 μl of 10 mM Tris.
SECTION: Size selection
12. Perform Pippin Prep size selection using 30 ul of the sample according to the instructions, keeping another 30 μl in case the size selection fails. You obtain 40 μl of the size selected sample. 
 
SECTION: Size selection
13. Measure concentration and check the profile of the sample using Fragment Analyzer/Tapestation/Bioanalyzer.
SECTION: Biotinylated fragment selection
14.  Mix well Dynabeads C1, make sure that all the beads are resuspended.
SECTION: Biotinylated fragment selection
15. Dispense 30 μl of beads in a PCR tube (for each capture).
SECTION: Biotinylated fragment selection
16. Wash 1:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 1 ml of 2x Bind and Wash buffer (10 mM Tris-HCl pH 7.5, 1 mM EDTA, 2 M NaCl).
 
SECTION: Biotinylated fragment selection
17. Wash 2:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 1 ml of 2x Bind and Wash buffer.
SECTION: Biotinylated fragment selection
18. Wash 3:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 40 μl of 2x Bind and Wash buffer.
SECTION: Biotinylated fragment selection
19. Combine 40 μl of the size-selected ligation product with 40 μl of the beads in 2x Bind and Wash buffer.
SECTION: Biotinylated fragment selection
20. Incubate 15 min at room temperature with gentle agitation. In the meantime, prepare the 1x wash buffer (see next step).

SECTION: Biotinylated fragment selection
21. For each sample, prepare 3 ml of 1x Bind and Wash buffer by mixing 1.5 ml of 2x Bind and Wash buffer and 1.5 ml of water.
SECTION: Biotinylated fragment selection
22. Wash 1:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 1 ml of 1x Bind and Wash buffer.
SECTION: Biotinylated fragment selection
23. Wash 2:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 1 ml of 1x Bind and Wash buffer.
SECTION: Biotinylated fragment selection
24. Wash 3:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 1 ml of 1x Bind and Wash buffer.
SECTION: Biotinylated fragment selection
25. Resuspend:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 30 μl of 10 mM Tris.
SECTION: PCR amplification
26. Prepare indexed primer solutions (see Before starting). Use different combination of indexed primers for each starting sample.
SECTION: PCR amplification
27. Prepare master mix 3 for 3 PCR reactions:
7.0 1
12.5 1
1.5 1
1.5 1
SECTION: PCR amplification
28. Mix 2.5 μl of the size selected ligation product with 22.5 μl of master mix 3 (total volume = 25 ul). Prepare 3 PCR reactions for each sample like this.
 

SECTION: PCR amplification
29. PCR program for KAPA HiFi HotStart ReadyMix:
3 min at 95°C
15 cycles* of:  
20 s at 98°C
15 s at 60°C
30 s at 72°C
5 min at 72°C
hold at 4°C.
 
* optimize the number of PCR cycles. Overamplification leads to heteroduplex formation, visible on a gel as secondary peaks/bands. Product like this will sequence normally but is harder to quantify. Too many PCR cycles also increase chimera formation and PCR error rate.
SECTION: PCR amplification
30. Pool the triplicates of each sample and perform AMPure cleanup with the beads:sample ratio 1:1 according to the manufacturer’s instructions. Elute in 20 μl of 10 mM Tris.
SECTION: PCR amplification
31. Quantify the library concentration using Qubit. Concentration around 10 ng/μl should be enough.
 
If not enough of probes is obtained, the rest of size-selected library can be amplified or the PCR product can be re-amplified: dilute the purified PCR product 10x and use in PCR reactions.
SECTION: PCR amplification
32. Pool the PCR products obtained from each sample together in equimolar ratios. 
 
Keep a small aliquot for verifying the restriction reaction (step 24) and aliqot for sequencing.
 
SECTION: Adapter removal
33. Prepare restriction digest reaction:
 
   reagent volume [μl]   water 39.75   CutSmart buffer (10x) 15.0   MseI (10,000 U/ml) 1.5   SbfI-HF (20,000 U/ml) 1.25   

SECTION: Adapter removal
34. Incubate for 3 hours at 37°C.
SECTION: Adapter removal
35. Check the restriction by running gel or Fragment Analyzer/Tapestation/Bioanalyzer on the sample before and after the restriction. There should be a visible peak of cut adapters and the main peak should shift accordingly.
SECTION: In vitro transcription of the libraries into RNA probes
36. In this step HiScribe T7 High Yield RNA Synthesis Kit is used. Usually 1 μl of the template yelds enough RNA. After the first reaction you can scale it up for the amount of probes needed.
 
Please make sure to read the HiScribeTM T7 Quick High Yield RNA Synthesis Kit Instruction Manual for instructions and troubleshooting.
SECTION: In vitro transcription of the libraries into RNA probes
37. Assemble the reaction:
   reagent volume [μl]   10X Reaction Buffer  3.0   ATP (100 mM) 3.0   GTP (100 mM) 3.0   CTP (100 mM) 3.0   UTP (100 mM) 2.0   Biotin-16-UTP (10 mM) 10.0   T7 RNA Polymerase Mix 3.0   

SECTION: In vitro transcription of the libraries into RNA probes
38. Incubate at 37°C overnight (up to 16 h).
SECTION: In vitro transcription of the libraries into RNA probes
39. Add 1 μl TURBO DNase (4 U) and incubate at 37°C for 30 minutes.
 
 
SECTION: In vitro transcription of the libraries into RNA probes
40. Purify the RNA using RNEasy Mini kit (Qiagen) using standard protocol except adding 665 μl EtOH to the RNA + RTL mix before loading on the column and elute in 30 μl of RNase-free water.


SECTION: In vitro transcription of the libraries into RNA probes
41. Quantify using Qubit RNA kit. You can check the RNA size using Fragment analyzer/Tapestation or by TBE/Urea gel electrophoresis.
SECTION: In vitro transcription of the libraries into RNA probes
42. Dilute the RNA probes to around 100 ng/μl.

Add 1 μl of SUPERase-In for each 19 μl of the probes.
Store at −80°C if not used immediately.
SECTION: Blocking RNA oligonucleotides synthesis
43. Anneal the oligonucleotides used as the template for T7 polymerase reaction. You need to anneal:
blocking RNA template for P5 adapter with T7 promoter,
blocking RNA template for P7 adapter with T7 promoter.

 
Heat the mix 1 min at 95°C, cool down to 10°C with ramp 0.1°C/s.

The annealed template can be stored long-term at -20°C.
SECTION: Blocking RNA oligonucleotides synthesis
44. Assemble the reaction for each annealed oligonucleotides pair:
   reagent volume [μl]   10X Reaction Buffer  3.0   ATP (100 mM) 3.0   GTP (100 mM) 3.0   CTP (100 mM) 3.0   UTP (100 mM) 2.0   Biotin-16-UTP (10 mM) 10.0   T7 RNA Polymerase Mix 3.0   

SECTION: Blocking RNA oligonucleotides synthesis
45. Incubate at 37°C overnight (up to 16 h).
SECTION: Blocking RNA oligonucleotides synthesis
46. Add 2 μl TURBO DNase (4 U) and incubate at 37°C for 30 minutes.
 
 
SECTION: Blocking RNA oligonucleotides synthesis
47. Purify the RNA using RNEasy Mini kit (Qiagen) using standard protocol except adding 665 μl EtOH to the RNA + RTL mix before loading on the column and elute in 30 μl of RNase-free water.


SECTION: Blocking RNA oligonucleotides synthesis
48. Quantify again using Qubit RNA kit. You can check the RNA size using Fragment analyzer/Tapestation or by TBE/Urea gel electrophoresis.
SECTION: Blocking RNA oligonucleotides synthesis
49. Adjust the concentration of each blocking RNA to 200 μM. This corresponds to the following concentrations:

BO.P5: 3129 ng/μl
BO.P5-multiplex: 4192 ng/μl
BO.P7-multiplex: 4366 ng/μl


Add 1 μl of SUPERase-In for each 19 μl of the final probes volume
 
Store at −80°C if not used immediately.

 

SECTION: Blocking RNA oligonucleotides synthesis
50. Mix the same amount of both blocking RNA oligonucleotides (BO.P5 and BO.P7-multiplex for single-indexed libraries or BO.P5-multiplex and BO.P7-multiplex for double-indexed libraries). The final concentration of each will be 100 μM.
SECTION: Hybridization reaction
51. Prepare the hybridization mix in the PCR tubes. Probes and blocking oligos are used in excess. So far, we tested the protocol using 500 ng of the genomic libraries and the same amount of the probes. For rare targets (aDNA for instance), you can increase library amount up to 2000 ng.
 
The below mixes are based on MyBaits protocol (http://www.mycroarray.com/pdf/MYbaits-manual-v3.pdf) and prepared in a bit larger quantities to account for evaporation and for easier handling.
 
Assemble the hybridization mix on ice:
 
   reagent volume [μl] final concentration   SSPE (20x) 9.0     EDTA (500 mM) 0.5     SDS (10%) 0.5     Denhardt’s solution (50x) 3.5     SUPERase-In (20 U/μL)* 3.5     hyRAD RNA probes (1000 ng)* 5.5     
 
*keep on ice 
 
Total volume = 20.0 μl, of which 18.0 μl is used in the hybridization.
 
SECTION: Hybridization reaction
52. 
 Prepare the blocking mix:
   reagent volume [μl]   Cot-1 DNA (1 mg/ml) 2.5   Salmon sperm DNA (1 mg/ml) 2.5    Mix of blocking RNA oligos 0.5   
Total volume = 5.5 μl, of which 5.0 μl si used in the hybridization.


 


SECTION: Hybridization reaction
53. Put 7 μl of the prepared Illumina library in the PCR tube (500 ng), add 5 μl of the blocking mix. Mix by pippeting.
SECTION: Hybridization reaction
54. Program the termocycler for two steps (both steps with a heated lid):
5 min at 95°C,
16-24 h at hybridization temperature (see below)

Usual hybridization temperature is 65°C. For rare targets like aDNA, incubation can last for up to 40 hours at lower temprature of 60 or 55°C.
SECTION: Hybridization reaction
55. Incubate the library + blocking mix 5 min at 95°C (1st step).
SECTION: Hybridization reaction
56. When thermocycler temperature decrease to the hybridization temperature (2nd step), put the tubes with the hybridization mix in the thermocycler, incubate the library and hybridization mix for 5 more minutes at 65°C.
SECTION: Hybridization reaction
57. Still at the hybridization temperature, add 18 μl of the hybridization mix to the library+blocking mix and mix by pippeting. Total volume is now 30 μl. Do not take the mixture out from the PCR machine to vortex or centrifuge at this step.
SECTION: Hybridization reaction
58. Incubate at the hybridization temperature for 16-24 hours (up to 40 hours and lower temperature for rare targets like aDNA).
SECTION: Preparation of Dynabeads
59.  Mix well Dynabeads C1, make sure that all the beads are resuspended.
SECTION: Preparation of Dynabeads
60. Dispense 30 μl of beads in a PCR tube (for each capture).
SECTION: Preparation of Dynabeads
61. Wash 1:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 200 μl of TEN.
 
SECTION: Preparation of Dynabeads
62. Wash 2:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 200 μl of TEN.
SECTION: Preparation of Dynabeads
63. Wash 3:
put on the magnetic rack, wait until the beads separate,
remove and discard supernatant,
take the tube off the magnetic rack,
resuspend in 70 μl of TEN.
SECTION: Preparation of Dynabeads
64. Store at room temperature until use.
SECTION: Washes
65. Prepare the aliquots of the wash buffers and Dynabeads in TEN and prewarm them to the hybridization temperature in a thermocycler.
SECTION: Washes
66. Keeping the hybridization reaction in a thermocycler at the hybridization temperature with the heated lid, add 70 ul of the Dynabeads in TEN to the 30 μl of the hybridization reaction and mix well by pipetting.
SECTION: Washes
67. Incubate for 30 min at the hybridization temperature in a thermocycler.
30 min
SECTION: Washes
68. Wash 1:
put on the magnetic rack, wait until the beads separate,
remove supernatant,
resuspend beads in 180 μl of 1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
mix well by vortexing, briefly centrifuge.
SECTION: Washes
69. Incubate for 15 min at the hybridization temperature in a thermocycler.
15 min
SECTION: Washes
70. Wash 2:
put on the magnetic rack, wait until the beads separate,
remove supernatant,
resuspend beads in 180 μl of 0.1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
mix well by vortexing, briefly centrifuge.
SECTION: Washes
71. Incubate for 10 min at the hybridization temperature in a thermocycler.
10 min
SECTION: Washes
72. Wash 3:
put on the magnetic rack, wait until the beads separate,
remove supernatant,
resuspend beads in 180 μl of 0.1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
mix well by vortexing, briefly centrifuge.
SECTION: Washes
73. Incubate for 10 min at the hybridization temperature in a thermocycler.
10 min
SECTION: Washes
74. Wash 4:
put on the magnetic rack, wait until the beads separate,
remove supernatant,
resuspend beads in 180 μl of 0.1x SSC / 0.1% SDS prewarmed to the hybridization temperature,
mix well by vortexing, briefly centrifuge.
SECTION: Washes
75. Incubate for 10 min at the hybridization temperature in a thermocycler.
10 min
SECTION: Washes
76. If you use Kapa HiFi Hot Start polymerase mix in the next steps you can keep the captured DNA on the beads and proceed to step 68.

Otherwise elute the DNA from the probes:
put on the magnetic rack, wait until the beads separate,
remove supernatant,
add 30 μl of 95°C 10 mM Tris,
mix well by pipetting.


SECTION: Washes
77. Incubate for 10 min, 95°C in a thermocycler.
5 min
SECTION: Washes
78. Put on the magnet; remove and retain the supernatant. 
This supernatant contains the hybridization-enriched products!
Discard the beads.
SECTION: Post-capture library amplification
79. Prepare re-amplification master mix. The volumes are per one capture-enriched library:
   reagent volume [μl]   water 14.1   Q5 polymerase buffer (5x) 10.0   dNTP mix (25 mM each) 0.4   PCR_F primer (5 μM) 5.0   PCR_R_post primer (5 μM) 5.0   Q5 hot-start DNA polymerase (2 U/μl) 0.5   
 
SECTION: Post-capture library amplification
80. Mix 42.5 μl of the master mix with 7.5 μl of the capture-enriched library.
SECTION: Post-capture library amplification
81. PCR program for KAPA HiFi HotStart ReadyMix:
3 min at 95°C
15 cycles* of:  
20 s at 98°C
15 s at 60°C
30 s at 72°C
5 min at 72°C
hold at 4°C.

* optimize the number of PCR cycles to obtain enough number of molecules for sequencing while using as few PCR cycles as possible.
SECTION: Post-capture library amplification
82. Purify with AMPure using beads:sample ratio 1:1, elute in 30 μl of 10 mM Tris.
SECTION: Post-capture library amplification
83. Verify the library profile and molarity using Fragment Analyzer/Tapestation/Bioanalyzer. Libraries can now be pooled in equimolar ratios.

Your libraries are now ready to be sent for sequencing, congratulations!