Sep 10, 2021
Tn5 based tagmentation library prep protocol, high throughput
- Charlotte Grace Sprehn1,
- Erik Enbody1,
- Yanjun Zan1,
- Leif Andersson1
- 1Uppsala University,Department of Medical Biochemistry and Microbiology, BMC Box 582, SE-751 24 Uppsala, Sweden.
- Erik Enbody: erik.enbody@gmail.com
- erikenbody
Protocol Citation: Charlotte Grace Sprehn, Erik Enbody, Yanjun Zan, Leif Andersson 2021. Tn5 based tagmentation library prep protocol, high throughput . protocols.io https://dx.doi.org/10.17504/protocols.io.bv5gn83w
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: June 26, 2021
Last Modified: September 10, 2021
Protocol Integer ID: 51080
Keywords: Tn5 based tagmentation library, Transposon Assembly, PCR Enrichment, Bead Cleanup,
Abstract
A very cost effective library preparation for whole-genome Illumina-based sequencing was previously demonstrated by by Picelli et al (Picelli et al., 2014). In this protocol, we optimized the Picelli et al protocol for very high throughput and is capable of generating libraries in 2x 96 well plates every 2 days. This protocol is adapted slightly from a protocol by Zan and Carlborg for high throughput library preparation (https://dx.doi.org/10.17504/protocols.io.rt8d6rw), but optimized for a project focused on high throughput of Darwin's finch samples (https://www.biorxiv.org/content/10.1101/2021.01.19.426595v1).
Attachments
du62bibpx.docx
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Guidelines
Appendix 1
1)2xTn5 dialysis Bf(DF): 1L (H20 added to vol.)
100 mM Hepes, pH 7.2 100 mL 1M or 23,83 g
200 mM NaCl 11.69 g NaCl
0.2 mM EDTA 400 uL 500 mM
2 mM DTT 2 mL 1M
0.2% Triton X-100 2 mL Triton X-100
20% Glycerol 252 g 100% Glycerol
2) 5X TAPS-MgCl2
50 mM TAPS-NaOH at pH 8.5, 25 mM MgCl2
Appendix 2
Tn5MErev, 5′-[phos]CTGTCTCTTATACACATCT-3′;
Tn5ME-A (Illumina FC-121-1030), 5′-TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG-3′;
Tn5ME-B (Illumina FC-121-1031), 5′-GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG-3′
Primer A = mix equal molar Tn5MErev with Tn5ME-A
Primer B = mix equal molar Tn5MErev with Tn5ME-B
PCR index primer are following Nextera XT Index Kit v2 - Index 2 (i5/i7) Adapters, sequences can be found on page 14 of illumina adapter sequences.You can either order the whole kit from illumine or synthesis it your self. I was using orders from IDT with standard desalting and it worked fine.
Materials
2xTn5 dialysis Bf(DF):
| A | B | |
| Hepes, pH 7.2 | 100 mM | |
| NaCl | 200 mM | |
| EDTA | 0.2 mM | |
| DTT | 2 mM | |
| Triton X-100 | 0.20% | |
| Glycerol | 20% |
5X TAPS-MgCl2:
50 mM TAPS-NaOH at pH 8.5, 25 mM MgCl2
Transposon Assembly
Transposon Assembly
2h 1m
2h 1m
Remove primers Tn5ME-A, Tn5ME-B, Tn5ME-Arev, Tn5ME-Brev, DF buffer, and Tn5 from freezer. Melt primers and buffer, mix by vortexing and spin. Leave Tn5 to melt on bench. Turn on 70 °C incubator.
Prepare primers A and B in 1.5 mL tube:
27.5 µL primer Tn5ME-A + 27.5 µL primer Tn5ME-rev = primer A (55 µL )
27.5 µL primer Tn5ME-B + 27.5 µL primer Tn5ME-rev = primer B (55 µL )
Incubate at 70 °C for 00:01:00 then place On ice .
1m
In a new 1.5 mL tube combine the following:
85 µL Tn5 (64 micromolar (µM) )
55 µL primer A
55 µL primer B
110 µL 2x DF buffer
Total305 µL
Mix by pipetting.
Incubate at Room temperature for 02:00:00 .
2h
Tagmentation
Tagmentation
2h 17m
2h 17m
Start thermocyclers so they are up to temp. Program: 55 °C 01:00:00 , 55 °C 01:00:00 for 15 µL volume.
2h
In new 2 mL tube add: (1x)
| A | B | |
| 900uL H2O | 4.5uL | |
| 400uL 5x TAPS buffer | 2uL | |
| 400uL 40% PEG | 2uL | |
| 200uL Tn5 mix | 1uL | |
| DNA (10ng/uL) | 1uL | |
| Total 1900uL mix |
Mix by inverting. Aliquot 225 µL into a strip of 8, and use strip to aliquot 9 µL into 2x 96-well plates using multichannel. If DNA is of questionable quality the volume can be increased to 1.5 or more, just scale water accordingly.
Move to DNA bench. Spin thawed DNA dilution plates. Add 1 µL DNA to prepared plates of mix. Seal with film and spin.
Incubate in thermocycler 00:10:00 at 55 °C .
10m
Remove from thermocycler. Carefully remove film and add 2.5 µL 0.2% SDS (prepared in a strip tube). Seal with new film and return to thermocyclers to incubate for 00:07:00 .
7m
PCR Enrichment
PCR Enrichment
7m
7m
Remove KAPA HiFi dNTPs and GC buffer from freezer and thaw, vortex, and spin. Index plates should be thawed and spun down.
Note
KAPA HiFi PCR enzyme should only be out of the freezer briefly.
In a 2 mL tube mix the following: (1x)
| A | B | |
| 576uL H2O | 3uL | |
| 960uL 5x PCR buffer | 5uL | |
| 57.6uL dNTPs (10mM) | 0.3uL | |
| 38.4uL HiFi PCR enzyme | 0.2uL | |
| Total 1632uL |
Mix by inverting and spin.
Aliquot 200 µL into an 8-strip. Move to DNA bench. Add 7.5 µL of mix to plates from tagmentation (which have 12.5 µL of product, so will end with 20 µL ).
Add index. Index plates are pre-prepared with equal amounts of both indices, so add 5 µL of the mix (=2.5uL each index) at 10 micromolar (µM) each.
Note
Be careful that plate positions match!
Cover PCR plates with lids (not film), spin, and place in thermocyclers for the following program:
When complete freeze at -20 °C Overnight or proceed to cleanup.
7m
Bead Cleanup
Bead Cleanup
1h 34m
1h 34m
Remove beads from cold room and let sit at Room temperature for 00:30:00 . Mix by inversion, light shaking before use. Pour about 2.6 mL into boat.
30m
Add 7.5 µL (with filter tips) to each well of PCR product. Cover with film and shake by hand until completely homogenous. Incubate at Room temperature 00:10:00 . Spin briefly.
10m
Place on magnet stands for 00:05:00 until all beads are on the sides.
5m
Pipette 28 µL to new plates. Pipette opposite the beads so as not to disturb them.
Add 4.5 µL beads (stir beads with pipette tip first). Cover with film, shake, and incubate 00:10:00 at Room temperature . Spin briefly.
10m
Place on magnet stand for 00:05:00 .
5m
While still on magnet, pipette out and throw away 22.5 µL of liquid-don’t touch beads. About 10 µL remains.
Make fresh 80% EtOH and add 70 µL to each. Place large stack of paper towels over the top and invert (towels, plate, and magnet) and shake. Leave upside down on towels for about a 00:01:00 .
1m
Repeat #24, but before dumping lift plate from magnet and allow all beads to drop to the bottoms of the tubes. Place back on magnets and leave for 00:03:00 before dumping. Shake vigorously and leave plate and magnet on its side on the bench to dry for about 00:03:00 . Let EtOH evaporate but do not leave too long, ensure no cracking in bead dot.
6m
Add 22 µL sterile H2O to each.
Cover with film, remove from magnets and shake by hand on trays vigorously. Spin briefly and put on shaker 00:02:00 @ 2000. Incubate at Room temperature 00:20:00 . Spin.
22m
Place on magnets for 00:05:00 . Prepare new plates during incubation.
5m
Remove film and pipette 19 µL into new plates. Seal with lids. Proceed to QC or place in cold room.
QC & Pool
QC & Pool
3m 30s
3m 30s
Use a fluorescence plate reader to quantify the amount of DNA in the two plates.
Note
We used a Tecan (Switzerland) plate reader and calibrated using a Qubit.
Pool accordingly for equimolar libraries. Store pools in cold room or freeze at -20 °C if not doing final wash within a day.
Note
Pool according to the required concentration needed for the sequencing platform. We usually made dilution plates to ~1.5ng/uL for Illumina NovaSeq applications, but changed slightly depending on the lowest concentration samples in the plates. Plan to lose ~half measurement from post-pooling wash.
Post-pooling wash
Post-pooling wash
1h 15m
1h 15m
Take beads out to bring to Room temperature 00:30:00 before starting. Mix thoroughly.
30m
Calculate how much of each pool you need to start with 1000 ng or as needed for your sequencing. Calculate how much beads you need for each pool (.45x and .3x).
Note
Adjust for how much you need to send for sequencing. You’ll end up with less than half of this starting concentration.
Transfer calculated amount of each pool into 2 mL tubes.
Add 0.45x AmpPure beads. Mix by gentle pipetting until homogenous.
Incubate 00:10:00 Room temperature . Put remaining pools back in cold room.
10m
Place tubes on tube magnet block for 00:05:00 . Label new 2 mL tubes.
5m
Transfer supernatant to new tubes. Pipette away from beads.
Add 0.3x beads. Mix until homogenous.
Incubate 00:10:00 at Room temperature .
10m
Place on magnet 00:05:00 .
5m
Pipette off supernatant. Leave tubes open on magnet.
Wash beads with 500 µL fresh 80% EtOH. Leave a couple seconds and pipette off EtOH into waste.
Let dry 00:05:00 at Room temperature . Aliquot some sterile H2O into a 2 mL tube.
5m
Suspend beads in 100 µL H2O, vortex well.
Place on magnet 00:10:00 .
10m
Move supernatant to new tubes.
Measure product on Qubit and Tapestation as desired. Adjust volumes to needed concentration via speedvac if needed. If too low can repeat this section with higher starting amount.