Mar 12, 2023
Metagenomic Library Prep from fecal sample lysate
- 1Arizona State University
Protocol Citation: Noah Noah Snyder-Mackler 2023. Metagenomic Library Prep from fecal sample lysate . protocols.io https://dx.doi.org/10.17504/protocols.io.rm7vzbob4vx1/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: In development
We are still developing and optimizing this protocol
Created: January 31, 2023
Last Modified: March 12, 2023
Protocol Integer ID: 76174
Keywords: Microbiome, Metagenome, Library, Lysate
Disclaimer
dual index sequences here
Abstract
Metagenomic library prep from fecal sample lysate using Illumina DNA prep kit (1/2 reactions).
Attachments
Guidelines
Abbreviations
BLT: Bead-Linked Transposomes
TB1: Tagmentation Buffer 1
MM: Master Mix
TWB: Tagmentation Wash Buffer
TSB: Tagmentation Stop Buffer
EPM: Enhanced PCR Mix
SPB: Sample Purification Beads
RSB: Resuspension Buffer
Materials
- PCR plates and covers
- Nuclease-Free water
- Illumina® DNA Prep (M) Tagmentation (24 Samples IPB)Illumina, Inc.Catalog #20060060
Bead-Linked Transposomes (BLT)
Tagmentation Buffer 1 (TB1)
Tagmentation Wash Buffer (TWB)
Tagmentation Stop Buffer (TSB)
Enhanced PCR Mix (EPM)
Sample Purification Beads (SPB)
Resuspension Buffer (RSB)
- Freshly prepared 80% ethanolFisher Scientific
- Index Adaptors (96)
- Magnetic stand
Preparation
Preparation
Add 18 µL of nuclease-free water and 2 µL of sample to a PCR plate
Tagmentation Reaction
Tagmentation Reaction
15m
15m
Take out TB1 and keep on On ice . Turn on thermocycler to 37 °C
Multiply by number of samples, for a 96 well plate use x100 and pipet mix together to create MM. Vortex BLT vigorously for 10 seconds to resuspend
| Reagent | x1 | x100 | |
| BLT | 5 ul | 500ul | |
| TB1 | 5 ul | 500ul |
Vortex and add 10 µL of reaction MM to each well
Note
We found this easiest using a repeater with a 0.5 mL tip for 50 aliquots of 10 µL
Pipette mix slowly 5 times using multichannel (should have 30 µL of volume in each well
Cover and seal plate. Then run "TAG" on thermocycler
- Choose the preheat lid option and set to 100 °C
- Set the reaction volume to 30 µL
- 55 °C for 00:15:00
- Hold at 10 °C
15m
During TAG incubation take out TSB and place at 37 °C and vortex before addition to samples
Add 10 µL of TSB to each sample well using repeater with 0.5 mL tip to stop enzymatic reaction
Note
Be careful because buffer is foamy and be sure to pipette mix after addition
Cover with thick plastic and run “PTC” protocol on thermocycler
- Choose the preheat lid option and set to 100 °C
- Set the reaction volume to 40 µL
- 55 °C for 00:15:00
- Hold at 10 °C
15m
Place on magnetic stand until beads pellet and discard supernatant
Note
Take out primer plates to thaw at 4 °C
- Wash 2x with 100 µL of TWB. Place on magnetic stand until beads pellet + remove and discard supernatant after each wash. After both washes add in another 100 µL of TWB to plate while on magnetic stand to prevent beads from drying while you prep PCR mix (don’t pipette mix)
Make PCR Master Mix
Make PCR Master Mix
1h 10m 15s
1h 10m 15s
Take out EPR and thaw on ice
- Make master mix, multiply by number of samples (use x100 for a 96 well plate) (use a 5 ml tube for a full plate of MM)
| Reagent | x1 | x100 | |
| EPR | 11 ul | 1,056 ul | |
| Nuclease free water | 11 ul | 1,056 ul |
Place on magnetic stand until beads pellet + remove and discard supernatant (TWB)
Add 20 µL of MM using repeater and 5 mL attachment
Add in 2.5 µL of i5 and i7 primers
Pipette mix, cover with thick plastic and run “BLT” 12 cycle on thermocycler ( 01:00:00 )
- Choose the preheat lid option and set to 100 °C
- 68 °C for 00:03:00
- 98 °C for 00:03:00
- 12 cycles of:
98 °C for 00:00:45
62 °C for 00:00:30
68 °C for 00:02:00
- 68 °C for 00:01:00
- Hold at 10 °C
Note
- Take out SPB and RSB while PCR is running to thaw
- You may need to place the RSB on the thermocycler at 37 °C to get it to thaw
1h 10m 15s
Bead Clean Up
Bead Clean Up
10m
10m
Grab a new PCR plate and add 60 µL of nuclease-free water using a multichannel pipette and 45 µL of SPB using a repeater with a .5 ml tip to each sample well
After PCR is complete spin down plate → place on magnetic stand → transfer 25 µL of supernatant to new PCR plate containing water and SPB. Discard old plate.
Pipette mix 10 times and incubate for 00:05:00 at Room temperature
During this incubation take out a new PCR plate and add 15 µL of SPB to each well using a repeater
5m
After the incubation place plate on magnetic stand until beads pellet
Transfer 125 µL to the new plate containing the 15 µL of SPB. Discard old plate.
Pipette mix 10 times and incubate for 00:05:00 at Room temperature
5m
Place on magnetic stand + remove and discard supernatant
Ethanol Washes
Ethanol Washes
3m 30s
3m 30s
Keep plate on magnetic stand and prepare fresh 80% ethanol
While keeping the plate on the magnetic stand add 200 µL of 80% ethanol without mixing
Incubate 00:00:30 + remove and discard supernatant. Repeat ethanol wash and discard supernatant.
Note
Use a 20 µL pipette to remove as much ethanol as possible
30s
Air dry while on magnetic stand (00:03:00 )
Note
be sure to not let beads crack
3m
Elution
Elution
2m
2m
Remove beads from stand and add 32 µL of RSB to beads
Pipette mix to resuspend and incubate at Room temperature for 00:02:00
2m
Place on magnetic stand and transfer supernatant to new and final sturdy PCR plate. Seal, label, and store!