Mar 12, 2023

Public workspaceMetagenomic Library Prep from fecal sample lysate 

Microbial Genomics
DOI
https://dx.doi.org/10.17504/protocols.io.rm7vzbob4vx1/v1
  • Noah Noah Snyder-Mackler1
  • 1Arizona State University
smacklabasu Snyder-Mackler
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DOI: https://dx.doi.org/10.17504/protocols.io.rm7vzbob4vx1/v1
External link: https://doi.org/10.1099/mgen.0.001595
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
Manuscript citation:
Saroff MJ, Haile AA, Baniel A, Kraberger S, Regney M, Harrach B, Kaján GL, Lu A, Beehner JC, Bergman TJ, Snyder-Mackler N, Varsani A, Schneider-Crease IA (2025) Early-life infection dynamics and genomic diversity of adenoviruses in a wild primate (Theropithecus gelada). Microbial Genomics 11(12). doi: 10.1099/mgen.0.001595
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, metagenomic library prep from fecal sample lysate, metagenomic library prep from fecal sample, metagenomic library prep, fecal sample lysate, using illumina dna prep kit, illumina dna prep kit, fecal sample
Disclaimer
dual index sequences here
Nextera 10bp dual index sequences
Abstract
Metagenomic library prep from fecal sample lysate using Illumina DNA prep kit (1/2 reactions).
Attachments
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18KB
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
  • ReagentIllumina® 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 Reagent80% ethanolFisher Scientific
  • Index Adaptors (96)
  • Magnetic stand



Troubleshooting
Preparation
Add Amount18 µL of nuclease-free water and Amount2 µL of sample to a PCR plate

Tagmentation Reaction
15m
Take out TB1 and keep on TemperatureOn ice . Turn on thermocycler to Temperature37 °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
Reagentx1x100
BLT5 ul 500ul
TB15 ul500ul

Vortex and add Amount10 µL of reaction MM to each well
Note
We found this easiest using a repeater with a Amount0.5 mL tip for 50 aliquots of Amount10 µL



Pipette mix slowly 5 times using multichannel (should have Amount30 µL of volume in each well
Cover and seal plate. Then run "TAG" on thermocycler
  • Choose the preheat lid option and set to Temperature100 °C
  • Set the reaction volume to Amount30 µL
  • Temperature55 °C for Duration00:15:00
  • Hold at Temperature10 °C
15m
During TAG incubation take out TSB and place at Temperature37 °C and vortex before addition to samples

Add Amount10 µL of TSB to each sample well using repeater with Amount0.5 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 Temperature100 °C
  • Set the reaction volume to Amount40 µL
  • Temperature55 °C for Duration00:15:00
  • Hold at Temperature10 °C
15m
Place on magnetic stand until beads pellet and discard supernatant
Note
Take out primer plates to thaw at Temperature4 °C


  1. Wash 2x with Amount100 µL of TWB. Place on magnetic stand until beads pellet + remove and discard supernatant after each wash. After both washes add in another Amount100 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
1h 10m 15s
Take out EPR and thaw on ice
  1. 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)
Reagentx1x100
EPR11 ul1,056 ul
Nuclease free water11 ul1,056 ul

Place on magnetic stand until beads pellet + remove and discard supernatant (TWB)
Add Amount20 µL of MM using repeater and Amount5 mL attachment

Add in Amount2.5 µL of i5 and i7 primers

Pipette mix, cover with thick plastic and run “BLT” 12 cycle on thermocycler ( Duration01:00:00 )
  • Choose the preheat lid option and set to Temperature100 °C
  • Temperature68 °C for Duration00:03:00
  • Temperature98 °C for Duration00:03:00
  • 12 cycles of:
Temperature98 °C for Duration00:00:45
Temperature62 °C for Duration00:00:30
Temperature68 °C for Duration00:02:00
  • Temperature68 °C for Duration00:01:00
  • Hold at Temperature10 °C

Note
  1. Take out SPB and RSB while PCR is running to thaw
  2. You may need to place the RSB on the thermocycler at Temperature37 °C to get it to thaw





1h 10m 15s
Bead Clean Up
10m
Grab a new PCR plate and add Amount60 µL of nuclease-free water using a multichannel pipette and Amount45 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 Amount25 µL of supernatant to new PCR plate containing water and SPB. Discard old plate.

Pipette mix 10 times and incubate for Duration00:05:00 at TemperatureRoom temperature
During this incubation take out a new PCR plate and add Amount15 µL of SPB to each well using a repeater
5m
After the incubation place plate on magnetic stand until beads pellet
Transfer Amount125 µL to the new plate containing the Amount15 µL of SPB. Discard old plate.

Pipette mix 10 times and incubate for Duration00:05:00 at TemperatureRoom temperature

5m
Place on magnetic stand + remove and discard supernatant
Ethanol Washes
3m 30s
Keep plate on magnetic stand and prepare fresh 80% ethanol
While keeping the plate on the magnetic stand add Amount200 µL of 80% ethanol without mixing

Incubate Duration00:00:30 + remove and discard supernatant. Repeat ethanol wash and discard supernatant.
Note
Use a Amount20 µL pipette to remove as much ethanol as possible



30s
Air dry while on magnetic stand (Duration00:03:00 )

Note
be sure to not let beads crack

3m
Elution
2m
Remove beads from stand and add Amount32 µL of RSB to beads

Pipette mix to resuspend and incubate at TemperatureRoom temperature for Duration00:02:00

2m
Place on magnetic stand and transfer supernatant to new and final sturdy PCR plate. Seal, label, and store!