TITLE: Automated Bar-Seq Library Preparation and Pooling
AUTHORS: David Ross, Nina Alperovich

[DESCRIPTION]
Protocol for automated Bar-Seq Library preparation

This protocol prepares 96 DNA samples, representing 24 samples from 4 different timepoints,  for multiplexed Illumina sequencing. The process starts with two rounds of PCR, each followed by a bead-based cleanup. The first round of PCR attaches primers that serve as tags to identify the timepoint and sample (e.g., time-points 1-4 in the Pooled, Growth-Based Assay Protocol). The second round of PCR attaches flow-cell adapters required for Illumina sequencing. Following the PCR and cleanup steps, the protocol outlines a procedure for pooling all samples together to ensure balanced representation during sequencing. This process starts with quantifying each sample's DNA concentration and then diluting and pooling together samples from the same timepoint.  The pooled samples' concentrations are then measured, and a small quantity of each timepoint sample is run on a gel to ensure the cleanup process was successful. The final step is to dilute and pool of all timepoint samples into one multiplexed tube for subsequent Illumina sequencing.

NOTES:
Before implementing this protocol, prepare the magnetic bead suspension following the Preparation of Sera-mag SpeedBeads protocol.
This protocol should be implemented after the Automation Protocol for Plasmid DNA Extraction from E. coli protocol
A fragment analyzer can also be used instead of a gel to determine if the PCR clean-up process was a success



[STEPS]
SECTION: Transfer Plasmid DNA to PCR Plate and Dilute with DI water
1. Pre-heat the on deck thermocycler (ODTC) for 1st PCR step 
SECTION: Transfer Plasmid DNA to PCR Plate and Dilute with DI water
2. Remove lids from PCR-Plate 1, Sample-Plate, and Reagent Plate
SECTION: Transfer Plasmid DNA to PCR Plate and Dilute with DI water
4. Transfer 35uL of extracted plasmid DNA to each well of PCR Plate 1
This step is done 24 samples (one time point) at a time, with user instructions to load each plasmid sample input plate (without lids) 
aspirate 1 mm below liquid surface, with liquid following ON
dispense 0.5 mm below liquid surface, with liquid following ON
SECTION: Transfer Plasmid DNA to PCR Plate and Dilute with DI water
3. Pipette 10uL nuclease-free water to each well in PCR plate 1
SECTION: Transfer Plasmid DNA to PCR Plate and Dilute with DI water
5. User instruction to remove plasmid sample input Sample-Plate 
SECTION: Run First PCR - using primers to identify samples from each timepoint
7. Add 28.125 uL Master Mix with forward primer for the appropriate timepoint and sample to each corresponding well of PCR plate 1
Mix 10x after dispensing
dispense 0.5 mm below liquid surface, with liquid following On
mix volume 70 uL
mixing speed 75 uL/s
mix position 1 mm (below dispense position)
SECTION: Run First PCR - using primers to identify samples from each timepoint
8. Put a PCR plate lid 1 on the PCR plate 1.
SECTION: Run First PCR - using primers to identify samples from each timepoint
6. Add 28.125 uL Master Mix with reverse primer for the appropriate timepoint and sample to each corresponding well of PCR plate 1
dispense 0.5 mm below liquid surface, with liquid following On
no mixing
SECTION: Run First PCR - using primers to identify samples from each timepoint
9. Move PCR-Plate 1 to ODTC 
SECTION: Run First PCR - using primers to identify samples from each timepoint
10. Run First PCR Rx using conditions (101.25 uL volume) using the following cycles:
98 C for 60 s 
3 cycles at:
98 C for 10 s 
58 C for 20 s 
72 C for 20 s 
3. 72 C for 60 s 
4. 23 C for 10 s
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
11. Pipette 54 uL magnetic bead suspension into each well of Midi plate 1
Bead ratio: 0.6x 
Mix the bead suspension 10x before pipetting
Get new tips for each well
Mix bead suspension again 2x before pipetting each well
Pipette bead suspension into Midi plate during the Step 1 PCR  
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
12. Move PCR plate 1 from ODTC; take lid off of PCR plate 1
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
13. Close ODTC; run PCR pre-heat method on ODTC (to get ready for Step 2 PCR) 
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
14. Shake Midi plate 1 with bead suspension for 10 second at 1800 RMP
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
15. Transfer 90 uL of each sample from PCR plate 1 to Midi plate 1
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
15.1. Mix 10x after dispensing
dispense 0.5 mm above bottom of well
mix volume 108 uL
mixing speed 100 uL/s
mix position 2 mm (below liquid surface)
liquid following ON
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
16. Incubate for 7 minutes at room temperature
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
17. Move Midi plate 1 to magnet base 
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
18. Wait for 4 minutes
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
20. Transfer 134 uL of supernatant from Midi plate 1 to Midi plate 2
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
21. Pipette 80.4 uL bead suspension to Midi plate 2 (on HHS)
Bead ratio: 0.6
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
21.1. Mix the bead suspension 10x before pipetting
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
21.2. Get new tips for each well and mix bead suspension again 2x before aspiration for each well
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
21.3. Mix 10x after dispensing
dispense 0 mm below liquid level 
10 mix cycles
mix volume 193 uL
mixing speed: 100 uL/s
mix position 1 mm (below liquid surface)
liquid following ON
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
43. Move Midi plate 3 to HHS (while 2nd PCR is running)
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
44. Pipette 89 uL bead suspension to Midi plate 3
Bead ratio 1.0x
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
44.1. Mix the bead suspension 10x before pipetting 
SECTION: Quantify the amount of input plasmid DNA
67. Measure the DNA concentrations (Cn) for all input plasmid samples using Qubit or an equivalent DNA quantitative measurement.
Note: The expected concentration (Cn's) for samples grown with zero selection antibiotic is typically around 0.5 ng/uL to 1 ng/uL, though samples from cultures grown with non-zero selection antibiotic may end up lower than that.
SECTION: Within each time point&#x27;s samples, calculate the samples&#x27; final pooling volumes (Vf)
70. Within each time point's samples (i.e., the 24 samples coming from each growth plate), identify the the four lowest-concentration samples.
SECTION: Within each time point&#x27;s samples, calculate the samples&#x27; final pooling volumes (Vf)
71. Calculate the mean of their concentrations in ng/µL, and define this concentration as the low concentration limit (Clow ) for that time point.
SECTION: Within each time point&#x27;s samples, calculate the samples&#x27; final pooling volumes (Vf)
72. For each sample, use the DNA concentration in ng/µL (Cn) and the following equation to calculate the pooling volume (Vn) for each sample:
Vn = Clow/(Cn + 0.1 ng/uL)*15 µL
SECTION: Within each time point&#x27;s samples, calculate the samples&#x27; final pooling volumes (Vf)
73. For each sample, set the final pooling volume (Vf) based on the following conditions: 
If Vn < 4 µL, set Vf = 4 µL
If Vn  > 15 µL, set Vf  = 15 µL.
SECTION: Within each time point, pool all samples and measure the pooled-DNA concentration
74. Within each time point's samples (i.e., the 24 samples coming each growth plate), pool together all samples into one container according to their final pooling volumes (Vf's) and store the remaining un-pooled samples at -20C.
Note: This will give one pooled sample for each time-point (four pooled samples in total), each at a concentration of approximately 20 ng/uL and with a volume of 100 to 300 uL. This is much more DNA than is needed for sequencing.
SECTION: Within each time point, pool all samples and measure the pooled-DNA concentration
76. For each of the four pooled samples, re-measure the DNA concentration and then store them at -20C until the next step can be accomplished.
SECTION: Dilute each time point&#x27;s sample and pool all samples for sequencing
77. To prepare samples for the sequencing provider, follow their instructions for pre-made Illumina libraries. 
Note: They usually give the sample requirements in molar concentration, typically 5 nmol/L, which is about 1 ng/uL for the BarSeq libraries. They usually require a minimum of 20 uL to 30 uL per lane of sequencing.
As an example, using the numbers above, a sample protocol might be to dilute 5 µL of each pooled sample into nuclease-free water to a concentration of 1 ng/uL. This will give much more than the minimum required volume, but it avoids the extra variability from pipetting smaller volumes.
SECTION: Dilute each time point&#x27;s sample and pool all samples for sequencing
78. Combine the diluted time point samples to be run on the sequencer. In the following manner:
For the first run: start with a single lane of NovaSeq, with all four time points combined in equal proportions. Then, after analyzing that data, if you need more data, order a second lane and send a second sample with only time points 2-4.
For subsequent runs: order a single lane of NovaSeq with the combine the time points with the following ratios: ⅓:1:1:1 for time points 1-4 (or ratios determined from first run).
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
44.2. Get new tip for each well & mix beads suspension again 2x before pipetting each well
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
45. Wait for PCR to finish 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
46. Move PCR plate 2 from ODTC; take lid off of PCR plate 2
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
47. Shake Midi plate 3 with bead suspension for 10 second at 1800 RMP
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
48. Transfer 89 uL from PCR plate 2 to Midi plate 3
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
48.1. Aspirate 0.5 mm above bottom of well with liquid following OFF
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
48.2. Mix 10x after dispensing
dispense 0.5 mm above bottom of well
mix volume 133.5 uL
mixing speed 100 uL/s
mix position 2 mm (below dispense position)
liquid following ON

SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
49. Incubate for 7 minutes
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
50. Move Midi plate 3 to magnet base 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
51. Wait for 5 minutes  
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
52. Remove and discard most of the supernatant (173 uL) 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
53. Pipette 200 uL 80% ethanol into Midi plate 4
jet dispense, from 20 mm height, using one tip for each row
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
56. Using smaller tips (e.g., 50 uL), remove remaining supernatant and dispose 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
55. Remove ethanol supernatant and discard 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
54. Wait 30 s 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
58. Wait 30 s to allow ethanol to finish drying 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
59. Pipette 50 uL Elution buffer into Midi plate 3
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
57. Move Midi plate 3 to HHS 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
59.1. Mix 2x after dispensing
dispense 1 mm above bottom of well
mix volume 37.5 uL
mix position 0 mm
liquid following OFF
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
60. Shake at 1800 RPM for 10 s
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
61. Shake at 400 RPM for 2 minutes
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
62. Incubate for 3 minutes without shaking
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
63. Move Midi plate 3 to magnet base 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
66. Replace lid on Output plate 
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
65. Transfer 45 uL supernatant from Midi plate 3 to Output plate
SECTION: Second PCR cleanup: Bind PCR product to beads and discard supernatant
64. Wait for 5 minutes 
Remove lid from Output plate during wait 
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
22. Incubate for 7 minutes at room temp, without shaking
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
24. Move Midi plate 2 to magnet base 
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
25. Wait for 5 minutes   
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
26. Remove and discard most of the supernatant (209.4 uL)
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
27. Pipette 200 uL 80% ethanol into Midi plate 2 (on magnet) 
Jet dispense, from 20 mm height, using one tip for each row
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
29. Using smaller tips (e.g., 50 uL), remove remaining supernatant and dispose 
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
28. Remove ethanol supernatant and discard 
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
30. Move Midi plate 2 to HHS
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
32.1. Mix 2x after dispensing
dispense 1 mm above bottom of well
mix volume 41.25 uL
mixing speed 100 uL/s
mix position 0 mm
liquid following OFF
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
32. Pipette 55 uL DI water into Midi plate 2
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
37. Start timer for 5 min incubation  
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
36. Move Midi plate 2 to magnet base 
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
35. Incubate for 3 minutes at room temp without shaking
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
34. Shake at 400 RPM for 2 minutes
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
33. Shake at 1800 RPM for 10 s
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
39. Transfer 45 uL of supernatant from Midi plate 2 to PCR plate 2 
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
37.1. While waiting, start next step by preparing the 2nd PCR reaction
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
39.1. Mix 10x after dispensing
dispense 0.5 mm below liquid surface
mix volume 69.3 uL
mixing speed 100 uL/s
mix position 1 mm (lower than dispense position)
liquid following ON
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
40. Put a PCR plate lid on PCR plate 2.
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
41. Move PCR-Plate 2 to ODTC 
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
42. Run PCR plate 2  (99 µL) using conditions:
98 C for 60 s 
15 Cycles at:
98 C for 10 s 
72 C for 30 s 
3. 72 C for 120 s 
4. 10 C for 20 s 

Note: during the 2nd PCR run, throw Midi plate 2 to waste and prepare Midi plate 3 following the instructions below.
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
38. Prepare PCR plate 2 by pipetting 54 uL of 2x master mix (containing both forward and reverse universal primers for Illumina sequencing) into each well
SECTION: Run 2nd PCR: using primers for Illumina multiplexing
38.1. Wait for 5 minute timer from the 1st PCR cleanup to finish.
SECTION: Quantify the amount of amplified DNA
69. The amount of amplified DNA should also be measured for each sample at each time point using Qubit or an equivalent DNA quantitative measurement. 
Expected yields are between 10 ng/uL and 30 ng/uL (in 45 uL), though some samples could have as little as 1 ng/uL (e.g., samples grown with the highest antibiotic concentration at the later time points).
SECTION: Run a gel for each time point
75. Run a gel for each time point, using the pooled product DNA for each, and compare the results with those obtained during BarSeq method development. 
The gels should not have any visible bands that are shorter than the expected amplicon size (315 bp)
Shorter bands indicate incomplete PCR cleanup that could result in a very low sequence count from Illumina sequencing. 
If the gels results look correct, proceed to the next step
SECTION: First PCR Cleanup Part 1: Bind template plasmid DNA to the beads and keep the supernatant
19. During 4 minute wait, move Midi Plate No. 2 to
Hamilton Heater Shaker (HHS).
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
23. During 7 minute wait, throw Midi Plate
No. 1 to waste
SECTION: First PCR Cleanup Part 2: bind PCR product to beads and discard the supernatant
31. Wait 30 s to allow ethanol to finish drying
SECTION: Run a gel for each sample from time point 1
68. Run a gel for each sample from time point 1, using the amplified product DNA for each, and compare the results with those obtained during BarSeq method development. 
The gels should not have any visible bands that are shorter than the expected amplicon size (315 bp)
Shorter bands indicate incomplete PCR cleanup that could result in a very low sequence count from Illumina sequencing. 
If the gels results look correct, proceed to the next step