TITLE: Primer ID MiSeq Library Prep for HIV-1 DR and diversity
AUTHORS: Shuntai Zhou

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">This is the protocol to prepare Primer ID MiSeq sequencing library. Viral RNA was first extracted using QIAamp viral RNA extraction kit.  The block of random nucleotides (Ns) in the cDNA primers served as the Primer ID. The Superscript III kit was used for the cDNA synthesis. We used two rounds of PCR to amplify the cDNA and incorporate Illumina indexed adapters with KAPA2G Robust and KAPA HiFi PCR kits, respectively. </div></div>

[STEPS]
?. [Prepare Primer Mix (Optional, only for multiplexed Primer ID library prep)]
For multiplexing sequencing, first, prepare Primer Mix.Example (For HIV drug resistance pipeline).   ABC1Regions
DR cDNA primer
DR F primer
2PR
R2614_PID
F2163AD
3RT
R3284_PID11
F2620_AD
4INR4752_PID11
F4383_AD
5V3
R7209_PID11V1F_AD

Make 10 µM primer mix: mix 10 µL of each primer in one set and 60 µL of dH2O. Primer Tables  AB1primer
sequence (5'-3')
2R2614_PID
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTTTAACTTTTGGGCCATCCATTCC
3R3284_PID11
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTCACTATAGGCTGTACTGTCCATTTATC
4R4752_PID11
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNATCGAATACTGCCATTTGTACTGC
5R7209_PID11
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTCCATTTTGCTYTAYTRABVTTACAATRTGC
6F2163AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTCAGAGCAGACCAGAGCCAACAGCCCCA
7F2620_AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNGGCCATTGACAGAAGAAAAAATAAAAGC
8F4383_AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNAAAAGGAGAAGCCATGCATG
9V1F_AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTTATGGGATCAAAGCCTAAAGCCATGTGTA
“N” in this Primer table is a random nucleotide.  AB1P1
AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTG
2Indexed Adapter
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTC
3ADPT_2a
GTGACTGGAGTTCAGACGTGTGCTC
The 6 “N”s are not random nucleotides. They are a set of 24 pre-designed indexed sequences.
ABC1Regions
DR cDNA primer
DR F primer
2PR
R2614_PID
F2163AD
3RT
R3284_PID11
F2620_AD
4INR4752_PID11
F4383_AD
5V3
R7209_PID11V1F_AD
AB1primer
sequence (5'-3')
2R2614_PID
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNCAGTTTAACTTTTGGGCCATCCATTCC
3R3284_PID11
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTCACTATAGGCTGTACTGTCCATTTATC
4R4752_PID11
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNATCGAATACTGCCATTTGTACTGC
5R7209_PID11
GTGACTGGAGTTCAGACGTGTGCTCTTCCGATCTNNNNNNNNNNNCAGTCCATTTTGCTYTAYTRABVTTACAATRTGC
6F2163AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTCAGAGCAGACCAGAGCCAACAGCCCCA
7F2620_AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNGGCCATTGACAGAAGAAAAAATAAAAGC
8F4383_AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNAAAAGGAGAAGCCATGCATG
9V1F_AD
GCCTCCCTCGCGCCATCAGAGATGTGTATAAGAGACAGNNNNTTATGGGATCAAAGCCTAAAGCCATGTGTA
AB1P1
AATGATACGGCGACCACCGAGATCTACACGCCTCCCTCGCGCCATCAGAGATGTG
2Indexed Adapter
CAAGCAGAAGACGGCATACGAGATNNNNNNGTGACTGGAGTTCAGACGTGTGCTC
3ADPT_2a
GTGACTGGAGTTCAGACGTGTGCTC
?. Mix well.
?. [cDNA synthesis ]
Pipette the following components into a 0.5 ml RNase-free tube:  ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
22.0dNTP Mix
10 mM each
0.5
&nbsp;
31.0cDNA primer
10 μM
0.25 μM
&nbsp;
423.0*RNA template
&nbsp;
&nbsp;
&nbsp;
526.00 Total volume
&nbsp;
&nbsp;
&nbsp;
ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
22.0dNTP Mix
10 mM each
0.5
&nbsp;
31.0cDNA primer
10 μM
0.25 μM
&nbsp;
423.0*RNA template
&nbsp;
&nbsp;
&nbsp;
526.00 Total volume
&nbsp;
&nbsp;
&nbsp;
[dNTPMix]
[cDNA primer]
[RNA template]
?. Place tube in 65°C heat block for 3-5’.
[65°C heat block]
?. Add the following components:  ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
28.05x buffer
5x
1x
&nbsp;
32.0DTT
100 mM
5
&nbsp;
42.0RNaseOUT
40 u/μl
2
&nbsp;
52.0SSIII RT
200 u/μl
10
&nbsp;
614.0 Per tube
&nbsp;
&nbsp;
&nbsp;
ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
28.05x buffer
5x
1x
&nbsp;
32.0DTT
100 mM
5
&nbsp;
42.0RNaseOUT
40 u/μl
2
&nbsp;
52.0SSIII RT
200 u/μl
10
&nbsp;
614.0 Per tube
&nbsp;
&nbsp;
&nbsp;
[5x buffer]
[DTT]
[RNaseOUT]
[SSII RT]
?. Mix and incubate at 50°C for 1 hr.
[Incubation at 50°C]
?. Increase to 55°C  and incubate for 1 hr.
[Incubation at 55°C]
?. Inactivate SSIII RT by heating at 70°C for 15’.
[Heating at 70°C]
?. To each tube, add 1 μl RNase H, incubate at 37°C for 20’.
[RNase H]
[Incubation at 37°C]
?. [Purification ]
Purify cDNA using Agencourt RNAClean XP. Resuspend the beads and take an aliquot out. Keep at room temperature for at least 30 minutes before use. (Should be in 1ml aliquots)
[Room temperature]
?. Transfer the cDNA reactions into 1.7 mL RNase-free tubes.
?. Resuspend the beads (Vortex). Add 28 µl of beads to 40 µl cDNA Agencourt RNAClean XP beads to each cDNA reaction.
[beads]
[cDNA]
?. Mix the Agencourt RNAClean XP and sample thoroughly by pipette mixing 15 times. No vortexing. Let the tube incubate at room temperature for 20 minutes before proceeding to the next step.
[Room temperature]
?. Place the tube onto the magnetic tube rack for 5 minutes to separate the beads from solution.
[Magnetic tube rack]
?. Slowly aspirate the cleared solution from the tube and discard. This step should be performed while the tube is situated on the rack. Do not disturb the magnetic beads, which have formed a spot on the side of the tube.
?. Dispense 400 μL of 70% ethanol into the tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard. (1/3) It is important to perform this step with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants.
[Incubation at room temperature]
?. Dispense 400 μL of 70% ethanol into the tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard. (2/3) It is important to perform this step with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants.
[Incubation at room temperature]
?. Let the reaction tube air-dry 10 minutes on the rack with the cap open. The tube(s) should air-dry until the last visible traces of ethanol evaporate. Over drying the sample may result in a lower recovery.
[Air-drying]
?. Remove the tube from the rack and resuspend beads in 40 µl DNase-free water by pipetting up and down, Place tube back on the rack and leave for 3 minutes.
[DNase-free water]
[Magnetic rack]
?. Remove the tube from the rack and resuspend beads in 24 µl DNase-free water by pipetting up and down. Place tube back on the rack and leave for 3 minutes.
[DNase-free water]
[Magnetic rack]
?. Pipette the eluant from the tube while it is situated on the magnetic tube rack.
?. [PCR 1]
Complete thaw and vortex KAPA reagents (except for enzyme) before use.
?. ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
210.0
5x Buffer A
5x
1x
&nbsp;
310.0
Enhancer
5x
1x
&nbsp;
41.0
dNTPs
10 mM
0.2 mM
&nbsp;
52.5
Forward primer
10 µM
0.5 µM
&nbsp;
62.5
ADPT_2a
10 µM
0.5 µM
&nbsp;
70.5
KAPA Robust polymerase
5 U/µl
2.5 U
&nbsp;
823.5
Template cDNA
&nbsp;
&nbsp;
&nbsp;
Prepare mastermix in cold box and use repeater pipette to add to each tube in the cold box. Add template cDNA to each tube and pipette up and down to mix.
ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
210.0
5x Buffer A
5x
1x
&nbsp;
310.0
Enhancer
5x
1x
&nbsp;
41.0
dNTPs
10 mM
0.2 mM
&nbsp;
52.5
Forward primer
10 µM
0.5 µM
&nbsp;
62.5
ADPT_2a
10 µM
0.5 µM
&nbsp;
70.5
KAPA Robust polymerase
5 U/µl
2.5 U
&nbsp;
823.5
Template cDNA
&nbsp;
&nbsp;
&nbsp;
[5x Buffer A]
[Enhancer]
[dNTPs]
[Forward primer]
[ADPT_2a]
[KAPA Robust polymerase]
[Template cDNA]
?. Cycle  AB195˚C
1 min
2&nbsp;
&nbsp;
395˚C
15 s
458˚C
1 min
572˚C
30 s
615-25 cycles
772˚C
3 min
84˚C
On hold
AB195˚C
1 min
2&nbsp;
&nbsp;
395˚C
15 s
458˚C
1 min
572˚C
30 s
615-25 cycles
772˚C
3 min
84˚C
On hold
?. [Purification PCR products]
Purify PCR products using AmpureXP PCR cleanup kits. Vortex the 1 ml aliquot and remove the needed volume. Keep at room temperature for at least 30 minutes before use.
[Room temperature]
?. Transfer the PCR1 reactions into 1.7 mL RNase-free tubes.
?. Resuspend the beads. Add 40 µl (Ratio: 0.6 – 0.8: 1, 36µl – 48µl) Ampure XP beads to each cDNA reaction.
[Ampure XP beads]
?. Mix the Ampure XP and sample thoroughly by vortexing. Let the tube incubate at room temperature for 5 minutes before proceeding to the next step (incube off the rack).
[Incubation at room temperature]
?. Place the tube onto the magnetic tube rack for 5 minutes to separate the beads from solution.
[Incubation magnetic rack]
?. Slowly aspirate the cleared solution from the tube and discard. This step should be performed while the tube is situated on the rack. Do not disturb the magnetic beads, which have formed a spot on the side of the tube.
?. Dispense 500 μL of 70% ethanol into the tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard. (1/2)
It is important to perform this step with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants.
[70% ethanol]
[Incubation at room temperature]
?. Dispense 500 μL of 70% ethanol into the tube and incubate for 30 seconds at room temperature. Aspirate out the ethanol and discard. (2/2)
It is important to perform this step with the tube situated on the rack. Do not disturb the separated magnetic beads. Be sure to remove all of the ethanol from the bottom of the well as it may contain residual contaminants.
[70% ethanol]
[Incubation at room temperature]
?. Let the reaction tube air-dry 10 minutes on the rack with the cap open. The tube(s) should air-dry until the last visible traces of ethanol evaporate. Over drying the sample may result in a lower recovery.
[Air-drying]
?. Remove the tube from the rack and resuspend beads in 50 µl DNase-free water by pipetting up and down. Place tube back on the rack and leave for 3 minutes.
[DNase-free water]
[Magnetic rack]
?. Pipette the 45 µl eluant from the tube while it is situated on the magnetic tube rack.
?. [PCR 2]
Complete thaw and vortex KAPA reagents (except for enzyme) before use.
?. ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
25.0
5x KAPA HiFi Fidelity Buffer
5x
1x
&nbsp;
31.0
dNTP Mix
10 mM
0.4 mM
&nbsp;
41.0
Uni Adapter (ADPT_P1)
10 µM
0.4 µM
&nbsp;
50.5
KAPA HiFI polymerase
1 U/µl
0.5 U
&nbsp;
61.0
Indexed Adapter
10 µM
0.4 µM
&nbsp;
72.0
Purified template DNA
&nbsp;
&nbsp;
&nbsp;
814.5
Water
&nbsp;
&nbsp;
&nbsp;
Prepare mastermix in cold box and use repeater pipette to add to each tube in the cold box. Add Indexed Adapter to each tube.
ABCDE1μl/tube
&nbsp;
[stock]
[final]
[mastermix]
25.0
5x KAPA HiFi Fidelity Buffer
5x
1x
&nbsp;
31.0
dNTP Mix
10 mM
0.4 mM
&nbsp;
41.0
Uni Adapter (ADPT_P1)
10 µM
0.4 µM
&nbsp;
50.5
KAPA HiFI polymerase
1 U/µl
0.5 U
&nbsp;
61.0
Indexed Adapter
10 µM
0.4 µM
&nbsp;
72.0
Purified template DNA
&nbsp;
&nbsp;
&nbsp;
814.5
Water
&nbsp;
&nbsp;
&nbsp;
[5x KAPA HiFi Fidelity Buffer]
[dNTP Mix]
[Uni Adapter (ADPT_P1)]
[KAPA HiFI polymerase]
[Indexed Adapter]
[Purified template DNA]
[Water]
?. Cycle (PCR machine #5 SZ -> ILM2):  AB195˚C
2 min
2&nbsp;
&nbsp;
398˚C
20 s
463˚C
15 s
572˚C
30 s
625 - 35 cycles
772˚C
3 min
84˚C
On hold
AB195˚C
2 min
2&nbsp;
&nbsp;
398˚C
20 s
463˚C
15 s
572˚C
30 s
625 - 35 cycles
772˚C
3 min
84˚C
On hold
?. [Gel Purification and quantification]
Before gel purification, run 2 µl products on 1% agarose gel to check the bands.
?. Gel purification.  (Qiagen QIAquick gel extraction kit)Run 2nd round PCR products on 1.2% agarose gel. E = 4 V/cm, T = 60 min.
?. Excise DNA fragment.
?. Weight the gel; add 3 volume of Buffer QG to 1 volume of gel.
?. Incubate at 50 ˚C for 10 minutes to completing dissolve. Vortex every 2-3 minutes to help dissolve.
[Incubation at 50 ˚C]
[Vortex]
?. Check the color of gel solution (should be yellow, otherwise add 10 µl 3M sodium acetate).
?. Place MinElute column, apply the sample to the column and centrifuge for 1 minute.
[Centrifugation]
?. Add 500 µl buffer QG and centrifuge for 1 minute.
[Buffer QG]
[Centrifugation]
?. Add 0.75 ml buffer PE, incubate for 5 minutes at room temperature, centrifuge for 1 minute.
[Incubation at room temperature]
[Centrifugation]
?. Discard the fluid, centrifuge for additional 3 minutes.
[Centrifugation]
?. Put the column in a new 1.7 ml tube, add 10 µl buffer EB. Stand for 4 minutes, centrifuge for 2 minutes.
[Stand]
?. Quantification using Invitrogen Qubit dsDNA BR Assay kit. See Qubit dsDNA BR assay protocol. Don’t use Nanodrop to quantify!
?. After quantification, pool libraries in equal amount. Use AMPure XP beads to purify pooled libraries (2:3, two washes), elute in 20 to 30 µL Elution Buffer.
?. Quantify the pooled library using Qubit dsDNA BR assay kit. Check the quality of the library by Agilent Bioanalyzer or Bio-rad Experion. Repeat purification if primer dimmer is present (~200bp).
?. Place the tube on ice for 1'.
[on ice]
?. Pipette the eluant from the tube while it is situated on the magnetic tube rack. Transfer the eluant into the tube with 28 µl of RNAcleanup XP beads.