Jan 12, 2026

Pre-Sequencing V.2

DOI
https://dx.doi.org/10.17504/protocols.io.j8nlk8kjxl5r/v2
  • 1University of Washington, Department of Genome Sciences, Brotman Baty Institute
Nahum Smith
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DOI: https://dx.doi.org/10.17504/protocols.io.j8nlk8kjxl5r/v2
Protocol Citation: Nahum Smith, Silvia Casadei 2026. Pre-Sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.j8nlk8kjxl5r/v2Version created by Nahum Smith
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: January 10, 2026
Last Modified: January 12, 2026
Protocol  Integer ID: 238342
Keywords: sequencing, protocol details the information, protocol detail, protocol, pre
Abstract
This protocol details the information about pre-sequencing.
Materials
Mastermix for PCR 1:

ABCDEFGHI
Sample d5 NCd5 R1D5 R2d5 R3d13 R1d13 R2d13 R3
DNA Quants (ng/uL) 152195210529784799613
Master Mix  x 1x 8x 8x 8x 8x 16x 16x 16
2X KAPA HiFi 25200200200200400400400
10um_F 1.512121212242424
10um_R1.512121212242424
DNA250 ng13.210.39.53.85.156.5
Waterto 25 uL162.8165.7166.5172.2346.9347345.5
Total50400400400400800800800
Mastermix for PCR 2:

AB
Master Mixx 1
2X KAPA HiFi12.5
10um_F (TrueSeqR1)0.75
10um_R (NexteraR2)0.75
water8.88
100x Sybr Green0.13
Total23
AMPured Rxn_12
25
Mastermix for PCR 3:
ABC
MasterMixx 1x29
2X KAPA HiFi (RM)12.5362.5
100x Sybr0.133.6
water7.38213.9
Total20580
10um_F (TrueSeq_P5)1.5
10um_R (Nextera_P7)1.5
AMPured Rxn_22
25
DNA and RNA isolation:
For all cell pellets harvested at the first timepoint (referred to as Day 05, or D5), isolate both DNA and RNA using the a modified QIAGEN AllPrep kit (see manufacturer instructions for : https://www.qiagen.com/us/resources/resourcedetail?id=580866a6-56c6-4674-8566-2852164d8519&lang=en).

(1) Determine amount of starting material (either cell count or cell pellet size) and adjust protocol using the tables below (Table 1a, 1b).
Table 1a. Amount of starting material for AllPrep DNA/RNA Mini

Cell pellet sizeNumber of cells
Very small < 0.1 x 10^6
Small 0.1 x 10^6 – 5 x 10^6
Medium 5 x 10^6 – 10 x 10^6
Large > 10 x 10^6

(2) Prepare Buffer RLT Plus w/1% β-MercaptoEthanol (β-ME). Determine volume of Buffer RLT Plus needed for the total number of samples and add 1% β-ME (Table 1b). Dispense β-ME in a fume hood.
Table 1b. Volumes of Buffer RLT Plus w/1%β-ME per starting material.

Cell pellet sizeNumber of harvested cellsVolume ofBuffer RLT / sampleVolume of β-ME / sample
Very small *< 0.1 x 10^6100ul1ul
Small 0.1 x 10^6 – 5 x 10^6350ul3.5ul
Medium 5 x 10^6 – 1 x 10^7600ul6ul
Large > 1 x 10^71000ul10ul

*For number of cells ≤0.1 x 10^6 (very small pellets), homogenization can be achieved by vortexing for 1 min without using a QIAshredder.
(3) Pipet up to 700μl of lysate directly into a QIAshredder spin column placed in a 2 ml collection tube, and centrifuge for 2 min at maximum speed. Save homogenized cell lysate in collection tube.
If processing >1x10^7 cells, apply volume to the same QIAshredder column. Collect homogenized cell lysate in a new collection tube, record lysate volumes, save homogenized cell lysates.
(Steps 1 thru 3 above replace STEP 1 of the AllPrep DNA/RNA Mini Kit Quick-Start Protocol, Part 1).
(4) Transfer the homogenized lysate to an AllPrep DNA spin column and centrifuge for 30s at ≥8000 x g (≥10,000 rpm). Place the AllPrep DNA spin column in a new 2 ml collection tube and keep column at RT for subsequent DNA purification. Proceed with the flow-through to RNA purification.
(^This is modified STEP 2 of the AllPrep DNA/RNA Mini Kit Quick-Start Protocol, Part 1).

(5) Total RNA purification. Add 1 volume of 70% ethanol to the flow-through. Mix well by pipetting. Do not centrifuge. Immediately transfer up to 700μl of the sample, including any precipitate, to an RNeasy spin column placed in a 2 ml collection tube. Centrifuge for 15 s at ≥8000 x g (≥10,000 rpm). Discard the flow-through. Save the collection tube for the following steps.

(6) Wash the RNA
  • Add 700μl Buffer RW1 to the RNeasy spin column. Close the lid and centrifuge for 15s at ≥8000 x g (≥10,000 rpm). Discard the flow-through. Save the collection tube.
  • Add 500μl Buffer RPE to the RNeasy spin column. Close the lid and centrifuge for 15s at ≥8000 x g (≥10,000 rpm). Discard the flow-through. Save the collection tube.
  • Add 500μl Buffer RPE to the RNeasy spin column. Close the lid and centrifuge for 2min at ≥8000 x g (≥10,000 rpm).
  • Place the RNeasy spin column in a new 2ml collection tube. Discard the old collection tube with the flow-through. Centrifuge at full speed for 1 min to dry the spin column membrane.

(7) Elute the RNA
Place the RNeasy spin column in a new 1.5ml Eppendorf tube. Add 50μl RNA Storage Solution (AM7001) directly to the spin column membrane. Close the lid gently, let the column sit for 1 min and centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the RNA. Using the 50uL eluate, repeat step 7. Reuse the 1.5ml Eppendorf tube. Centrifuge for 1 min at ≥8000 x g (≥10,000 rpm).
(^These are modified STEPS 1-6 of the AllPrep DNA/RNA Mini Kit Quick-Start Protocol, Part 2, Total RNA purification)

(8) Genomic DNA purification
Return to the AllPrep DNA spin column left at RT.

(9) Wash the genomic DNA
  • Add 500 μl Buffer AW1 to the AllPrep DNA spin column in 2 ml collection tube. Close the lid gently, and centrifuge for 15 s at ≥8000 x g (≥10,000 rpm) to wash the spin column membrane. Discard the flow-through. Reuse the collection tube.
  • Add 500 μl Buffer AW2 to the AllPrep DNA spin column. Close the lid gently, and centrifuge for 2 min at full speed to wash the spin column membrane.
  • Place the AllPrep DNA spin column in a new 2ml collection tube. Discard the old collection tube with the flow-through. Centrifuge at full speed for 1 min to dry the spin column membrane.
  • Incubate the AllPrep DNA spin column with lid open at room temperature (15–25°C) for 2 mins.

(10) Elute the genomic DNA
Place the AllPrep DNA spin column in a new 1.5ml Eppendorf tube. Add 50μl of Nuclease-treated water directly to the spin column membrane and close the lid. Incubate at room temperature (15–25°C) for 1 min. Centrifuge for 1 min at ≥8000 x g (≥10,000 rpm) to elute the DNA. Using the 50uL eluate, repeat step 10. Reuse the 1.5ml Eppendorf tube. Centrifuge for 1 min at ≥8000 x g (≥10,000 rpm).
(^These are modified STEPS 1-3 of the AllPrep DNA/RNA Mini Kit Quick-Start Protocol, Part 2, Genomic DNA purification).




  • Resuspend all DNA samples in50 µL of Nuclease free water.

PreSequencing Amplification:
Library prep from each DNA sample requires three consecutive PCRs:

  • PCR1 or selection PCR leveraging genomic primers specifically designed to amplify the SNV library from DNA integrated in HAP1 genomes, and not from library plasmid DNA.

  • PCR2, or target specific PCR, uses PCR1 amplicons as template to amplify a sequencing amplicon spanning the SNV library.

  • PCR3, or index PCR, uses universal adapters in the sequencing amplicon as handles to link dual sequencing indices.

PreSequencing PCR1 or selection PCR:

Note
Gradient PCR: using unedited HAP-1 DNA as template, perform an 8-temperature gradient PCR (Tm range 58 °C -68 °C ) to determine the optimal PCR conditions for each PCR1 primer pair. Set up gradient PCRs in10 µL reaction volume with 50 ng total DNA.
PreSequencing PCR1: Multiple technical replicates of each DNA sample are amplified in PCR1.

  • For each Day 5 DNA sample, set up to amplify 8 replicates in independent PCR wells.

  • For each Day 13, Day 17 and/or Day 21 samples set up to amplify 16 replicates in independent PCR wells.

Since cell populations are generally larger at later timepoints, an increased number of PCR reactions is used to minimize jackpotting and sufficiently sample the cell population.


Prepare one MasterMix per DNA to be amplified with enough reagents for the total number of replicates; include DNA in the matsermix and dispense equally in wells (8 for D5 samples, 16 for D13, D17 or D21 samples).

ABCDEFGHI
Sample d5 NCd5 R1D5 R2d5 R3d13 R1d13 R2d13 R3
DNA Quants (ng/uL) 152195210529784799613
Master Mix  x 1x 8x 8x 8x 8x 16x 16x 16
2X KAPA HiFi 25200200200200400400400
10um_F 1.512121212242424
10um_R1.512121212242424
DNA250 ng13.210.39.53.85.156.5
Waterto 25 uL162.8165.7166.5172.2346.9347345.5
Total50400400400400800800800
Example table depicting mastermix volumes of Negative Control, 3 biological replicates of Day 05 harvests, and 3 biological replicates of Day 13 harvests.
ABC
STEPTEMPTIME
Initial Denaturation95°C4 minutes
10-20 Cycles*98°C30 seconds
(tbd)°C15 seconds
72°C40 seconds
72°C4 minutes
Hold4°Co/n
*Do not over-amplify, end PCR at inflection point before reaching plateau.


Carry out PCR at optimal Tm and cycle number (based on gradient PCR parameters), do not over-amplify.
Pool 1/5 volume from each PCR sample (e.g. 10 µL of 50 µL reaction) and merge in an Eppendorf tube.

Clean with 1.2x AMPure beads followed by two 80% EtOH washes and resuspend in initial volume of water (resuspend in 80 µL water if pooling 10 µL from each of the 8 PCR replicates and 160 µL water if 16 PCR replicates).

Verify amplicon size on a 2% agarose gel in TAE buffer (120V, 00:00:00 ).

PreSequencing PCR2: amplify the specific target use sequencing primers to link universal adapters to the sequencing amplicon.

Set up a MasterMix for the total number of samples (PCR1 replicates have been merged) in25 µL reaction volume, adding DNA (AMPured PCR1) independently to each PCR reaction.

AB
Master Mixx 1
2X KAPA HiFi12.5
10um_F (TrueSeqR1)0.75
10um_R (NexteraR2)0.75
water8.88
100x Sybr Green0.13
Total23
AMPured Rxn_12
25
ABC
STEPTEMPTIME
Initial Denaturation98°C3 minutes
10-20 Cycles (TBD)*98°C20 seconds
60°C15 seconds
72°C15 seconds
Hold4°Co/n
*Do not over-amplify, end PCR at inflection point before reaching plateau.


Perform PCR2 following standard parameters (Tm 60 °C ); abort reaction at amplification inflection before reaching amplification plateau.

Clean with 1.2 x AMPure beads and resuspend in 100 µL volume.

Use 2 µL as template for PCR3.

PreSequencing PCR3: amplify the sequencing amplicon from PCR2 to add sequencing indices.
Prepare a MasterMix for the total number of samples to be amplified; add primers and DNA (AMPured PCR2) independently to each well.

ABC
MasterMixx 1x29
2X KAPA HiFi (RM)12.5362.5
100x Sybr0.133.6
water7.38213.9
Total20580
10um_F (TrueSeq_P5)1.5
10um_R (Nextera_P7)1.5
AMPured Rxn_22
25
ABC
STEPTEMPTIME
Initial Denaturation98°C30 seconds
10-20 Cycles (TBD)**98°C10 seconds
66°C15 seconds
72°C15 seconds
Hold4°Co/n
*Do not over-amplify, though make sure to obtain enough library for pooling.

Clean with 1.2 x AMPure beads and resuspend in 25 µL volume.

Check PCR2 and PCR3 on a 2% agarose gel; if loaded next to each other, it should be possible to observe the larger size of the PCR3 amplicon due to the sequencing indices.
Prepare a 1:10 dilution of the AMPured PCR3.
Using a Qubit High Sensitivity DNA kit, or similar, determine the concentration of each sample and dilute each library to2 µL .

In an Eppendorf tube, pool equal volumes (e.g., 5 µL ) of each 2 µL library for sequencing.

Check concentration using a Qubit High Sensitivity DNA kit.