Page Table of Contents

Base Protocol

Full details of the protocol: 2 -step Library preparation protocol for 16s or ITS

1. 3 ul of 0.01 pg/ul cross contamination oligos and 0.03 pg/ul synthgene added to 15 ul of each sample.

2. Concentration measured via absorption and normalized to 10 ng/ul via automated liquid handler.

3. Two stage PCR performed with MagBead Cleanups in between.  Duplicate stage 1 PCR performed for each sample DNA and loci. First Stage Primers amplify regions of interest, add unique pairs of sequence barcoding, and a portion of the Illumina adaptor. Second stage completes Illumina adaptor addition.

4. After second magbead cleanup, amplicon product concentrations were measured via absorption. Products were relatively similar concentrations, with an order of magnitude. So, 2 ul of each sample was combined across 8 tubes. The 8 tubes were vortexed and spun before 50 ul of each was combined for the pool.

5. Finally, most amplifications showing low concentration via final absorption were run singly at a 1:500 dilution on qPCR along with the diluted full pool in quadruplicate. 2 out of the 64 individual samples did not show amplification.

Identifying Your Samples

<your plate name>: 16S barcode plate 1, 16S barcode plate 2, ITS barcode plate 1, ITS barcode plate 2

Plate KG1: 16S0E4, 16S0F4, ITS0A6, ITS0B6

Plate KG2: 16S0H4, 16S0G4, ITS0C6, ITS0D6

Plate KG3: 16S0A7, 16S0B7, ITS0E6, ITS0F6

Plate KG4: 16S0C7, 16S0D7, ITS0G6, IT0H6

Plate KG5: 16S0E7 (Column 1 and 2), ITS0A7 (Column 1 and 2)*

*I used 2 aliquots of Zymo’s Mock Community DNA as positive controls in wells G1 and H1 of KG5

Sample to Barcode Pair Maps:

These are also available in Google Sheets: https://docs.google.com/spreadsheets/d/122dymtIe6QtoU6GZzoJvTaFG0J85O5fBKrOFZXVOCFU/edit?usp=sharing

Quality Management

1. All final individual amplicon preps were measured via absorption.

2. qPCR Results – 1:500 dilutions of 8 samples from each full plate amplification were amplified via qPCR (KAPA Library Quant Kit Illumina ROX Low). Samples were selected semi randomly with a bias toward low final absorption readings. Two low absorption samples failed to amplify. All others showed amplification.

Lab Notes

16S Library Prep:

Add 7 ul Master mix to each well:

Add 6 ul 0.25 uM paired primers

KG1: 16S0E4, 16S0F4, ITS0A6, ITS0B6

KG2: 16S0H4, 16S0G4, ITS0C6, ITS0D6

KG3: 16S0A7, 16S0B7, ITS0E6, ITS0F6

KG4: 16S0C7, 16S0D7, ITS0G6, IT0H6

KG5: 16S0E7 (Column 1 and 2), ITS0A7 (Column 1 and 2)

Add 2 ul of appropriate gDNA to each well

Run on Thermocycler Program 35GSAF1:

Pool Duplicates

Purify samples using modified manual AxyPrep MagBead PCR Clean-Up (GSAF uses AmpPure XP):

AmpPure XP PCR cleanup and AxyPrep MagBead have the exact same stock protocol!

Equilibrate Beads to room Temperature

Add 24 ul of MagBeads to each well; Pipette mix up and down 10 times.

Incubate at RT for 5 minutes

Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Allow plate to air dry for 7 minutes.

Remove sample plate from magnet plate.

Add 15 ul TE per GSAF protocol; pipette mix 10+ times

Incubate at RT for 2 minutes

Place sample plate back on magnet for 5 minutes or until all wells are cleared.

Prepare next MasterMix while sample plate is on the magnet plate.

Add 5 ul FlowCell MasterMix to new plate:

Transfer 10 ul from plate on magnet plate to new strip tubes.

Run on Thermocycler Program 35GSAF2:

Purify samples using GSAF’s second modified MagBead protocol:

Equilibrate Beads to room Temperature

Add 15 ul H2O to each sample

Add 24 ul (0.8 x 60 ul) of MagBeads to each well; Pipette mix up and down 10 times

Incubate at RT for 5 minutes

Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Allow plate to air dry for 7 minutes.

Remove sample plate from magnet plate.

Add 23 ul TE per GSAF protocol; pipette mix 10 times

Incubate at RT for 2 minutes

Place sample plate back on magnet for 5 minutes or until all wells are cleared.

Transfer 20 ul to a clean PCR plate.

____________________________________________________________________________________________________________

ITS Library Prep:

Swap out KG4 G9’s blank for Mock Community positive control

Add 12 ul Master mix to each well:

Add 6 ul 0.25 uM paired primers ____________________

Add 2 ul of appropriate gDNA to each well

Run on Thermocycler Program 35GSAF1:

Pool Duplicates

Purify samples using modified manual AxyPrep MagBead PCR Clean-Up (GSAF uses AmpPure XP):

AmpPure XP PCR cleanup and AxyPrep MagBead have the exact same stock protocol!

Equilibrate Beads to room Temperature

Add 24 ul of MagBeads to each well; Pipette mix up and down 10 times.

Incubate at RT for 5 minutes

Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Allow plate to air dry for 7 minutes.

Remove sample plate from magnet plate.

Add 15 ul TE per GSAF protocol; pipette mix 10+ times

Incubate at RT for 2 minutes

Place sample plate back on magnet for 5 minutes or until all wells are cleared.

Prepare next MasterMix while sample plate is on the magnet plate.

Add 20 ul FlowCell MasterMix to new plate:

Transfer 10 ul from plate on magnet plate to new strip tubes.

Run on Thermocycler Program 35GSAF2:

Purify samples using GSAF’s second modified MagBead protocol:

Equilibrate Beads to room Temperature

Add 15 ul H2O to each sample

Add 24 ul (0.8 x 60 ul) of MagBeads to each well; Pipette mix up and down 10 times

Incubate at RT for 5 minutes

Secure plate on magnet plate; incubate at RT for 5 minutes (until wells are clear)

Remove 54 ul from each well; keep tips to left or right depending on the column to avoid bead pellet.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Add 100 ul Fresh 80% EtOH to each well. Incubate 30 seconds. Remove 100 ul from each well.

Allow plate to air dry for 7 minutes.

Remove sample plate from magnet plate.

Add 23 ul TE per GSAF protocol; pipette mix 10 times

Incubate at RT for 2 minutes

Place sample plate back on magnet for 5 minutes or until all wells are cleared.

Transfer 20 ul to a clean PCR plate.