Sep 13, 2024
Low-cost Tagmentation Library Prep for low-coverage Illumina sequencing
- 1University of Georgia
Protocol Citation: Matthew Farnitano, Andrea Sweigart 2024. Low-cost Tagmentation Library Prep for low-coverage Illumina sequencing. protocols.io https://dx.doi.org/10.17504/protocols.io.6qpvr8zrplmk/v1
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: September 11, 2024
Last Modified: September 13, 2024
Protocol Integer ID: 107434
Keywords: Library prep, Illumina, Low-coverage sequencing, Tagmentation
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Abstract
Tagmentation is a popular approach for preparing Illumina sequencing libraries, but kits can be pricey. Using a bulk-prepped and pre-loaded Tn5 tagmentation enzyme, this protocol is a simple, low-cost and high-throughput procedure for prepping libraries. It is designed for multiplexed low-coverage whole genome sequencing, a useful method for genotyping of many samples at low per-sample cost. Samples are prepped in 96-well PCR plates and combined into multiplexed batches. We have successfully used this protocol with Mimulus guttatus and Mimulus nasutus wild-collected and greenhouse-grown samples.
Before you start
Before you start
Obtain Tn5 tagmentation enzyme, pre-loaded with universal adapter sequences. Enzyme purification and loading guidelines are not provided here: see reference Lu et al. (2017).
Quantify input DNA. We recommend 96-well fluorescence quantification with a plate reader and a kit such as the Invitrogen Quant-iT dsDNA Assay kit, Catalog #Q33120.
Dilute DNA to approximately 1 ng/uL per sample in molecular-grade H2O. The required input is 1 uL of diluted DNA; we have had success with less than 1 ng on some samples, but final read coverage is correlated with initial sample concentration. If sample concentration is already relatively low (<5 ng/uL), we typically do not dilute. We dilute samples in batches of similar concentration, erring on the side of higher final concentrations. For example, all samples at initial concentration of 5-15 ng/uL might be diluted by 1/5.
Before starting tagmentation, pull out the following to thaw:
- Tagmentation (TD) buffer
- Taq Master Mix (for PCR step)
- i5 and i7 indices (for PCR step) -- prepare working dilutions if necessary
i5 indices are in individual tubes at 100 uM -- they do not need to be diluted (optionally, dilute to 10uM). Use a separate i5 index for each plate of 96 samples, one index per plate.
i7 indices are in a 96-well plate, the stock plate is at 100uM. Make a working dilution plate of 10uM.
Use this same i7 plate for all sample plates, one well per sample.
NOTE: our i5 and i7 index adapter sequences are included in attached files to this protocol.
Tagmentation
Tagmentation
Set up tagmentation reaction
Prepare a 96-well PCR plate with 1 uL diluted DNA (~1 ng/uL) per sample well
Create reagent Master Mix:
| A | B | C | |
| Reagent | Amount per sample | Amount per 96-well plate | |
| H2O | 3.497 uL | 349.7 uL | |
| TD Buffer | 0.42 uL | 42 uL | |
| Tn5 enzyme | 0.083 uL | 8.3 uL |
Add 4 uL Master Mix to each well of diluted DNA in 96-well PCR plate (final volume 5 uL)
Run tagmentation reaction in thermocycler:
| A | B | C | |
| Step | Duration | Temperature | |
| 1 | 15 mins | 55C | |
| 2 | Hold | 10C |
Once temperature has reached 10C, quickly move to the next step. Keep the reaction on ice!
PCR
PCR
Set up PCR Reaction
**Samples and PCR reagents should remain on ice as much as possible during PCR setup**
Prepare PCR Master Mix (one mix per plate):
| A | B | C | |
| Reagent | Amount per sample | Amount per 96-well plate | |
| OneTaq Hot Start 2X Master Mix* | 12.5 uL | 1350 uL | |
| H2O | 6.95 uL | 750.6 uL | |
| i5 index adapter, 100uM stock** | 0.05 uL | 5.4 uL |
*OneTaq Hot Start 2X Master Mix, New England Biolabs, Catalog #M0484L
**Optionally, dilute stock to 10uM and add 10X listed volume (adjust H2O amounts accordingly)
Add 0.5 uL i7 adapters, 10uM, (96-well plate format) to each tagmentation sample well using multichannel pipet
Add 19.5 uL PCR Master Mix to each tagmentation sample well.
To allow for multichannel pipetting, either move PCR Master Mix to a thin trough, or divide evenly into 8-count strip tubes, 263uL per row of 12 samples.
Run PCR reaction in thermocycler:
| A | B | C | |
| Step | Duration | Temperature | |
| 1 | 3 mins | 72 C | |
| 2 | 1 min | 95 C | |
| 3: | Repeat x18 | ||
| 3a | 10 sec | 95 C | |
| 3b | 20 sec | 55 C | |
| 3c | 3 mins | 72 C | |
| 4 | 5 mins | 72 C | |
| 5 | Hold | 4 C |
After PCR, plates can be held at 4C overnight if necessary. If leaving overnight, do NOT combine samples yet! Once combined, proceed immediately to bead cleanup.
Pooling and bead cleanup
Pooling and bead cleanup
Bring SPRI magnetic beads* to room temperature for cleanup, and resuspend fully using a vortexer.
*We have used "CleanNGS DNA & RNA Cleanup Magnetic Beads" from Bulldawg Bio, Catalog #CNGS050
Pool 48 samples (cols 1-6, or cols 7-12 from one plate) into a single batch for cleanup.
Pipette 20 uL from each sample well into a single microcentrifuge tube (~960 uL total)
Mix well with a P1000 pipet and transfer half (~480 uL) to a second tube. Proceed with cleanup of both tubes. You will have two tubes per 48-sample batch (4 tubes per 96-well plate).
Use SPRI magnetic beads and magnetic tube rack to clean up PCR products.
Add 1.5 times sample volume of SPRI beads to each tube (720 uL SPRI beads per 480 uL sample tube).
Gently pipet up and down 5-10 times to mix thoroughly.
Incubate 5 mins at room temperature on tabletop.
Place tubes on magnetic tube rack. Wait 5 mins. Beads should collect on side of tube, leaving a clear supernatant. Your DNA will be bound to the magnetic beads at this point.
**WHILE TUBES REMAIN ON MAGNET:
Discard clear supernatant with a pipet, taking care not to disturb beads
**WHILE TUBES REMAIN ON MAGNET:
Pipet 500 uL fresh 70% Ethanol directly onto beads. Let sit for 1 min. (Wash #1)
**WHILE TUBES REMAIN ON MAGNET:
Discard clear supernatant with a pipet, taking care not to disturb beads
**WHILE TUBES REMAIN ON MAGNET:
Pipet 500 uL fresh 70% Ethanol directly onto beads. Let sit for 1 min. (Wash #2)
**WHILE TUBES REMAIN ON MAGNET:
Discard clear supernatant with a pipet, taking care not to disturb beads
**WHILE TUBES REMAIN ON MAGNET:
Let beads air-dry until they have a matte appearance, with slight cracking. If necessary, use a P20 or P200 to remove excess liquid drops without disturbing beads.
Once dry, remove tubes from magnet.
Pipet 17 uL molecular-grade H2O directly onto beads. Gently pipet liquid back onto beads repeatedly, massaging the beads until they resuspend. You should end up with a single aliquot of uniform brown liquid at the bottom of the tube, with no beads stuck to the side.
Incubate 3 mins at room temperature on tabletop.
Place tubes back onto the magnetic tube rack. Wait 5 minutes for beads to collect on the side of the tube, leaving a clear supernatant. YOUR DNA IS NOW IN THE CLEAR SUPERNATANT.
**WHILE TUBES REMAIN ON MAGNET:
Carefully pipet ~15 uL of the clear supernatant, which contains your DNA, into a fresh labelled tube. Do not disturb or pipet up any of the brown beads. You may need to leave a small amount (1-2 uL) behind with the beads. If the beads are accidentally disturbed, return the liquid to the tube and let the beads settle onto the magnet again before re-trying.
At this point, the two replicate tubes from each 48-sample batch can be combined, for a final volume of 30 uL per batch of 48 samples.
The library prep is now complete. We use a Qubit fluorometer to measure the concentration of each 48-sample batch, then combine equal ng amounts into a single tube to submit for sequencing.
Protocol references
Lu, Z., Hofmeister, B. T., Vollmers, C., DuBois, R. M., & Schmitz, R. J. (2017). Combining ATAC-seq with nuclei sorting for discovery of cis-regulatory regions in plant genomes. Nucleic Acids Research, 45(6), e41. https://doi.org/10.1093/nar/gkw1179