TITLE: Automation Protocol for DNA Barcode Sequencing Library Preparation
AUTHORS: Nina Alperovich,  Drew S Tack,  Olga Vasilyeva,  Sasha F Levy, David Ross

[DESCRIPTION]
<div class = "text-blocks"><div class = "text-block">Here, we describe a protocol for automated library preparation for DNA barcode sequencing. The protocol uses a Hamilton NGS-STAR automated liquid handler equipped with an automated thermocycler.</div><div class = "text-block">This protocol requires an automated liquid handler (Hamilton Robotics, NGS-STAR) equipped with an automated thermocycler.</div></div>

[STEPS]
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Add 9 g PEG-8000 to a new 50 mL sterile conical tube.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Add 10 mL 5 mol/L NaCl.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Add 500 µL 1 mol/L Tris-HCl, pH8.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Add 500 µL 0.5 mol/L EDTA.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Fill 50 mL tube to 45 mL using sterile nuclease free water (vendor), mix by vortexing every 1-2 minutes until PEG goes into solution.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Add 27.5 µL Tween-20 and mix gently by inverting 4-6 times.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Vortex Sera-mag SpeedBeads to resuspend and transfer 1 mL to each of two 2 mL micro-centrifuge tubes.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Place SpeedBeads solutions on a magnetic separation rack until beads are drawn to the magnet and solutions are clear.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Remove supernatant.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Add 1 mL 1x TE Buffer (Thermo Scientific, cat. #12090015 pH 8.0) to each micro-centrifuge tube containing beads, remove from magnetic stand, and mix by vortexing.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Repeated steps 8-10 two additional times.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
After mixing, add both 1 mL SpeedBeads solutions to the 50 mL conical tube containing the PEG/NaCl solution.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Fill conical tube to the 50 mL mark with sterile nuclease free water, and gently mix until solution is a uniformed brown color.
?. [Preparation of Magnetic Bead/PEG-NaCl Stock]
Wrap in foil (or place in dark container) and store at 4 °C until use.
?. [Preparation of DNA barcode sequencing samples]
Transfer 35 µL of each plasmid DNA sample to a PCR plate (Bio-Rad, HSP9645).
?. [Preparation of DNA barcode sequencing samples]
Add 5 µL ApaI Restriction Enzyme Solution for plasmid DNA linearization; mix 3x by repeated aspiration and dispense.
?. [Preparation of DNA barcode sequencing samples]
Incubate samples in PCR plate in automated thermocycler at 37 °C for 15 minutes.
?. [Preparation of DNA barcode sequencing samples]
Add 2.5 µL Forward Index Primer and 2.5 µL Reverse Index Primer to each sample (20 µmol/L stock primer solutions, all primer sequences are listed in supplementary files). The Forward and Reverse Index Primers attached sample multiplexing tags to the resulting amplicons so that the different samples could be distinguished when they are pooled and run on the same sequencing flow cell. Eight different Forward Index Primers and 12 different Reverse Index Primers are used to uniquely label the amplicons from each sample across 24 different chemical environments and four time points used for barcode sequencing.
?. [Preparation of DNA barcode sequencing samples]
Add 45 µL 2x PCR Master Mix 1 (Thermo Scientific Phusion Flash High-Fidelity PCR Master Mix, Cat. #F548L) to each sample; mix 3x by repeated aspiration and dispense.
?. [Preparation of DNA barcode sequencing samples]
Run the first PCR reaction in automated thermocycler with the following conditions:    a. Initial denaturation: 98 °C for 60 s    b. 3 cycles:         i.   Denaturation: 98 °C for 10 s         ii.  Annealing: 58 °C for 20 s         iii. Elongation: 72 °C for 20 s     c. Final Extension: 72 °C for 60 s     d. Cooling: 20 °C for 15 s
?. [Preparation of DNA barcode sequencing samples]
During the first PCR reaction, pipette 48 µL Magnetic Bead/PEG-NaCl Stock into each of 24 wells in a 96-well midi plate (Abgene, AB-0765); mix bead stock well by repeated aspiration and dispense before each transfer.
?. [Preparation of DNA barcode sequencing samples]
When the first PCR reaction is finished, transfer 80 µL from each PCR reaction to a well in the midi plate; mix PCR reaction solution and bead stock 10x by repeated aspiration and dispense; wait for 5 minutes. During this step, the ratio of Bead Stock to PCR reaction volume is 0.6x. Consequently, because of the relatively low PEG concentration in the mixture, the 5.5kb plasmid template binds to the beads.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate to 96-well magnet base to pull the magnetic beads out of suspension; wait for 2 minutes.
?. [Preparation of DNA barcode sequencing samples]
Transfer 128 µL of supernatant from each sample to a clean well in the midi plate (still on the magnet base); wait an additional 3 minutes for final bead separation.
?. [Preparation of DNA barcode sequencing samples]
Transfer 118 µL of supernatant (containing the 218 bp PCR amplicon and primers)from each sample to another clean well in the midi plate; move the midi plate from the magnet base to automated shaker.
?. [Preparation of DNA barcode sequencing samples]
Add 70.8 µL Magnetic Bead Stock (0.6x of 118 µL supernatant volume) to each sample; mix supernatant and bead stock 10x by repeated aspiration and dispense; wait for 5 minutes. During this step, only the 206 bp PCR amplicon binds to the beads; the 70 bp primers remain in the supernatant.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate back to the magnet base; wait for 5 minutes.
?. [Preparation of DNA barcode sequencing samples]
Remove and discard the supernatant from each well.
?. [Preparation of DNA barcode sequencing samples]
Add 200 µL 80 % ethanol to each magnetic bead pellet; wait 30 s.
?. [Preparation of DNA barcode sequencing samples]
Remove and discard the ethanol supernatant; then, using 50 µL tips, remove residual supernatant from the bottom of each well.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate from magnet base to the automated shaker; allow magnetic bead pellets to dry for 5 minutes at room temperature.
?. [Preparation of DNA barcode sequencing samples]
Add 35 µL nuclease-free water to each sample; resuspend beads by 5x repeated aspiration and dispense.
?. [Preparation of DNA barcode sequencing samples]
Mix samples by shaking at 1800 rpm for 10 s; wait for 5 minutes.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate back to the magnet base; wait for 5 minutes.
?. [Preparation of DNA barcode sequencing samples]
During 5-minute wait, pipette 36 µL PCR Master Mix 2 (containing primers) into each of 24 wells in a PCR plate.
?. [Preparation of DNA barcode sequencing samples]
After 5-minute wait, transfer 30 µL of each supernatant from the midi plate (on magnet base) to the PCR plate.
?. [Preparation of DNA barcode sequencing samples]
Run the second PCR reaction in the automated thermocycler with the following conditions:    a. Initial denaturation: 98 °C for 60 s    b. 15 cycles:         i.  Denaturation: 98 °C for 10 s         ii. Annealing and Elongation: 72 °C for 30 s     c. Final Extension: 72 °C for 120 s     d. Cooling: 15 °C for 20 s
?. [Preparation of DNA barcode sequencing samples]
During the second PCR reaction, pipette 66 µL (1.1x of the 60 µL PCR volume) Magnetic Bead Stock into each of 24 clean wells in the midi plate (on the automated shaker); mix bead stock well by repeated aspiration and dispense before each transfer.
?. [Preparation of DNA barcode sequencing samples]
When the second PCR reaction is finished, transfer 60 µL from each PCR reaction to a well in the midi plate; mix the PCR reaction solution and bead stock 10x by repeated aspiration and dispensing; wait for 5 minutes. During this step, with a Bead Stock to PCR reaction volume ratio of 1.1, the 306 bp PCR amplicon binds to the beads while the 70 bp primers remain in the supernatant.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate back to the magnet base; wait for 4 minutes.
?. [Preparation of DNA barcode sequencing samples]
Remove and discard the supernatant from each well.
?. [Preparation of DNA barcode sequencing samples]
Add 200 µL 80 % ethanol to each magnetic bead pellet; wait 30 s.
?. [Preparation of DNA barcode sequencing samples]
Remove and discard the ethanol supernatant; then, using 50 µL tips, remove residual supernatant from bottom of each well.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate from the magnet base to the automated shaker; allow magnetic bead pellets to dry for 5 minutes at room temperature.
?. [Preparation of DNA barcode sequencing samples]
Add 50 µL Elution Buffer to each sample; resuspend beads by 5x repeated aspiration and dispense.
?. [Preparation of DNA barcode sequencing samples]
Mix samples by shaking at 1800 rpm for 10 s; wait for 5 minutes.
?. [Preparation of DNA barcode sequencing samples]
Move the midi plate back to the magnet base; wait for 5 minutes.
?. [Preparation of DNA barcode sequencing samples]
Transfer the supernatant to pooled sequencing sample, one pooled sample per input time point.
?. [Preparation of DNA barcode sequencing samples]
Transfer pooled sample to a 2 mL micro-centrifuge tube.
?. [Preparation of DNA barcode sequencing samples]
Place the tube on a magnetic separation rack until the remaining beads are drawn to the magnet (approximately 5 minutes). Transferred the fully clarified samples to a new 2 mL micro-centrifuge tube and store them at 4 °C until needed for sequencing.