-
Notifications
You must be signed in to change notification settings - Fork 3
Experimental protocols
The forward and reverse 24-nucleotide barcode sequences are given in Tables S1 and S2; the full-length barcode linked forward and reverse primers for pET22b(+) are given in Tables S3 and S4. If using pET22B(+) as a cloning vector, the sequences listed in Tables S3 and S4 can be directly used for ordering primers at 100 µM concentrations from commercial suppliers. If using a different cloning vector, the user must verify that the primers in the “Primer Design” section would still amplify the desired region.
There are 96 unique forward barcode-linked primers, corresponding to each well of a 96-well plate; there are 96 reverse barcode-linked primers, corresponding to the ability to sequence 96 different plates as a unique reverse barcode is used for each plate. The barcode-linked primers are listed in the tables below (Tables S3 and S4); the 96 F primers and 96 R primers are both ordered in plate format at 100 µM concentration.
Each well sequenced using LevSeq is encoded by a unique combination of a forward (F) and a reverse (R) barcode. The reverse barcode would identify the sequence to a specific plate, and the forward barcode would identify the sequence to a specific well. Since these barcode-linked primers are compatible with any gene cloned in a specific cloning vector, it is convenient to keep a set of barcode-linked primer plates on hand.
We used the same eight barcode plates throughout this work, and they are named LevSeq01–LevSeq08. The exact barcodes used are given in Tables S5–12. The LevSeq software assumes the forward barcodes used for library preparation are laid out in the order given in Tables S5 and S12. To build the barcode plates depicted in Tables S5–12, we followed the below procedure:
1. A 10-fold dilution of the 100 µM forward barcode-linked primer plate from IDT was prepared by adding 10 µL of the forward primer stock to 90 µL ddH2O to a final concentration of 10 µM, keeping the well layout constant. Dilutions were performed in half skirted PCR plates (BIO-RAD HSP9601).
2. A 10-fold dilution of the reverse barcode-linked primer from IDT was prepared by adding 150 µL of the 100 µM reverse primer stock to 1350 µL ddH2O to a final concentration of 10 µM. Dilutions were performed in Eppendorf tubes.
3. To create plates for sequencing of forward/reverse primer mixes, 80 µL ddH2O were added to each well of each of the eight plates. Then, 10 µL diluted (10 µM) forward barcode plate was aliquoted to each well keeping the layout constant.
4. A unique reverse barcode was added to every well of each of the eight plates. To do so, 10 µL of diluted (10 µM) reverse barcode from step 2 was used. For instance, LevSeq01 has forward barcode 01 – 96 and reverse barcode 01, LevSeq02 has forward barcode 01 – 96 and reverse barcode 02, and so on. The final concentration of each barcode plate is 1 µM each for the forward and reverse primers (10X stock).
5. Eight of the LevSeq barcode plates (LevSeq 01 – 08) at 10X stock concentration were made in step 4. In order to make 1X reaction-ready barcode plates, the following dilution was performed: To another set of eight fully skirted PCR plates, 90 µL of ddH2O were added, followed by 10 µL of diluted LevSeq barcode stock plates (1 µM). The final concentration of each ready-to-use barcode plate is 0.1 µM for both forward and reverse barcode linked primers.
6. When not in use, the 10X stocks prepared in step 4 were stored at -20 °C, while the barcode plates were stored at 4 °C. The 100 µM, 10 µM, and 1 µM barcode plates can be stored for long periods of time.
The steps below can be followed to complete a LevSeq sample preparation ready for loading onto an Oxford Nanopore flow cell (FLO-MIN114). Note that when designing a new set of barcode-linked backbone-specific primers, it is recommended to test the primers and PCR protocol using a few wells before deploying them for plate-scale reactions. The library protocol below provides part numbers that are for the materials and reagents used while developing this protocol, reagents from other providers should work as well.
1. Prepare a PCR master mix for the number of plates to be sequenced according to the table below.
| Component | Amount per plate (µL) |
| ThermopoI Buffer (NEB B9004L) | 144 |
| 10 mM dNTPs (NEB N0447) | 28.8 |
| Taq Polymerase (NEB M0267) | 7.2 |
| Mol-Bio Grade DMSO (mp 194819) | 57.6 |
| ddH2O | 770 |
2. Add 7 µL of master mix to each well of as many half-skirted PCR plates (USA scientific 1402-9700) as will be sequenced. These are referred to as “PCR plates”.
3. Stamp 2 µL of the 1-µM barcode-linked primer mix from the barcode plates into the PCR plates.
a. “Stamp” means “apply to all wells, keeping the plate layout consistent”.
4. Stamp 1 µL of overnight culture from each plate to be sequenced into the PCR plates. Record which barcode plate was used with which PCR plate.
5. Complete a PCR using the below thermal cycler program. This colony PCR amplifies the entire gene of interest from the template DNA contained in the cell culture.
| Step | Temperature (°C) | Time |
| 1 | 95 | 5 min |
| 2 | 95 | 20 s |
| 3 | TD 68 -> 63.5 | 20 s |
| 4 | 68 | 1 min per kb |
| 5 | Return to 2, 9x | |
| 6 | 95 | 20 s |
| 7 | 68 | 1 min per kb |
| 8 | Return to 6, 24x | |
| 9 | 68 | 5 min |
| 10 | 4 | Hold |
a. “TD” in step 3 stands for “touchdown”, a touchdown step decreases the temperature each cycle. The TD in the above PCR starts at 68 °C and drops to 63.5 °C by the end, decreasing by 0.5 °C per cycle.
b. Extension time in steps 4 and 7 are recommended to be minimally 1 minute per kb of gene, a 2-min extension time was used for genes below 1 kb during development of this protocol.
6. While the PCR is running, prepare a 1% agarose gel with SYBR gold added (Thermo Fisher Scientific, S11494).
7. Once the PCR is completed, for each plate, pool 5 µL of each reaction into an Eppendorf tube, for a total of 480 µL of pooled PCR products per plate. Pooling will leave you with as many Eppendorf tubes as you have plates.
a. Note: Pooling can be performed using a 12-channel multichannel pipette. 1) First, transfer 5 µL of reactions from each row in the PCR plate (to be sequenced) into a new row of a 96-well PCR plate. Since there are eight rows in the original plate, each well of the row in the new 96-well PCR plate will have 40 µL of pooled PCR products. 2) Transfer 30 µL from each well in the single row of pooled reactions using a single-channel pipette to a microcentrifuge tube. Since there are 12 columns in the new 96-well PCR plate, the microcentrifuge tube should have 360 µL of pooled PCR products that contain PCR reactions of every well of the PCR plate (to be sequenced).
b. Alternatively, a liquid handling robot can be used for this task. It is important that 5 µL from each well of each PCR plate is combined into one microcentrifuge tube to improve the evenness of sequencing coverage. There will now be an equal number of microcentrifuge tubes to as plates being sequenced.
8. For each tube made in step 7, take 100 µL of pooled PCR reactions and add it to 20 µL 6x loading dye (NEB B7025S) in a microcentrifuge tube. The remaining pooled reaction can be stored at -20 °C for future use.
9. Load the contents of each tube made in step 9 into the agarose gel prepared in step 7. Each tube should have separated lanes. Load a 10 µL of 1 kb ladder in the flanking lanes.
10. Run the agarose gel at 120 V until the bands have sufficiently migrated. Reference the ladder to identify the PCR products which should be the size of the gene plus about 100 bp. The extra bases come from the barcodes and the region between the primer attachment sites and the open reading frame.
11. Gel extract the desired bands. Samples from separated PCR reaction plates should have their own microcentrifuge tube to hold the extracted gels. Commercial kits such as the Zymoclean Gel DNA Recovery Kit (Zymo Research, D4001) are typically used for this step. Elution should be performed using 10 µL of ddH2O rather than elution buffer.
12. After gel extraction, measure the DNA concentration of each gel-extracted PCR product from each plate. We use a GE NanoVue Plus to measure DNA concentration in ng/µL unit.
13. Combine the gel-extracted PCR products from each plate in equimolar concentrations. In general, 200 ng of pooled DNA sample per 1-kb gene are sufficient for any sample preparation. For experiments that use the same length template, the weight is directly proportional to molarity. Hence the same amount of DNA in nanograms from each plate can be combined into one microcentrifuge tube. For example, if plate one after gel extraction has a measured concentration of 20 ng/µL and plate two has a concentration of 10 ng/µL, then to make 20 µL of 10 ng/µL final sample, 5 µL of the first plate and 10 µL of the second plate were added to 5 µL of ddH2O to make the final sample.
14. After step 13, there should be one single tube of cleaned, normalized DNA consisting of all genes from all plates to be sequenced. Depending on the Oxford Nanopore sample preparation protocol, this sample is adjusted to the desired concentration before sample preparation for sequencing.
15. A MinION flow cell with the LSK114 ligation sequencing kit was used while developing this protocol, which recommends 200 fmol of amplicon DNA for sample preparation. This translates to 120 ng for a 1-kb gene.
16. From the concentration-normalized sample prepared in step 13, transfer the equivalent volume of 120 ng of pooled DNA into a new microcentrifuge tube. This pooled DNA sample (120 ng) will be carried forward for Oxford Nanopore sequencing sample preparation. Protocols are linked here
17. Once the sample is loaded onto the flow cell for sequencing, we recommend choosing the super accurate basecalling option to ensure retrieval of the highest quality sequences while setting up the sequencing run. It is recommended to stop the flow cell after the basecalled bases reach a data volume of 0.02 Gb per plate to be sequenced, so an experiment with 20 plates would be stopped after collecting 0.4 Gb of basecalled bases.