Template Information:

Air and Brew samples collected and extracted in Weinig lab and delivered in plate labeled Brew_20_Nov

Initial Sample Prep:

• Vortex and quick spin plate Brew_20_Nov

• Add 30 ul TE to fill out the last columns and treat as samples.

• Vortex and spin control oligo pool (16S and ITS coligo 0.01 pg/ul each AND 16S SG_GR and ITS SG_GR 0.03 pg/ul each.

• Add 6 ul control oligo plate to the sample aliquot plate in a well to well fashion (A1 to A1, B1 to B1, . . .) This can be done using an 8 channel or the 96 channel. If you use the 8 channel, you can reuse the dome caps on the control plate as long as you avoid contamination. Return control plate. Label side opposite numbers of aliquot plate with an asterisk.

• Seal, vortex, and spin aliquot plate.

• Check concentration on Synergy HTX and save file on petalibrary as plate name. Make a new folder under DNA Quantification labeled “iSeqPilot1”. Add a circle around the asterisk when concentrations have been measured. This plate can go on the top shelf of the left hand refrigerator well sealed.

• Only one sample was above 10 ng/ul. Hand dilute it to 10 ng/ul

  • Add 52.8 ul TE to Well G2

PCR1:

• Create MasterMix:

• Add 7 ul to each well of 4 hard shell, full skirt plates, Seal with tape seals, rub with gray sealer across all wells twice, and then around the edge, store in refrigerator until needed labeled “PCR1”

• Spin down 4 “PCR1” plates. Vortex and spin 1 Brew_20_Nov plate. Vortex and spin two 16S MID plates and 2 ITS MID plates.

• Remove the seals from the 4 “PCR1” plates. Attach tips from a freshly opened 20 ul tip box TO THE BENCHSMART. Add 2 ul to all “PCR1” plates.

• Dispose of these tips.

• Grab 1 new 20 ul tip box. Cover 3 of the “PCR1” plates. Grab one of the MID plates.

• Remove the bubble caps from the appropriate MID plate.

• Transfer 6 ul to the corresponding column of the “PCR1” plate. Recap the MID plate and cap the resulting plate.

• Repeat the previous 4 steps matching each “PCR1” plate with a unique MID plate.

• Move “_Norm” plate to -80 storage. Move MID plates to “Used MIDs” storage section.

• Record Mid Plates here:

• Spin down resulting “PCR1”s and then add to thermocyclers running “35GSAF1”

PCR2 Mastermix Preparation:

• Add 5 ul master mix to all wells of 2 hard shell full skirted plates. Seal with tape seals and store in refrigerator until needed

Intermediate Cleanup:

The majority of this was done on the Nimbus platform using “AxyPrep MagBead PCR1 No MM”

Manually, it was done:

• Equilibrate Beads to room Temperature

• Add 24 ul of MagBeads to each well and 15 ul of replicate to same well of other replicate (match loci)

• 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 65 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

• Reaspirate from each well to assure maximum EtOH removal

• Allow plate to air dry for 7 minutes.

• Remove sample plate from magnet plate.

• Add 40 ul TE; pipette mix 10+ times. Incubate 2 minutes at RT.

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

• Transfer 40 ul to labeled transparent plate (Plate1 PCR1 MIDPlate1 MIDPlate2)

• Transfer 10 ul from transparent plate to “PCR2” plate with Mastermix already added.

• Label Plate1 PCR2 MIDPlate1 MIDPlate2

• Seal “PCR2”s with bubble strips and run on thermocycler 35GSAF2 program

Final Cleanup:

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 40 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 40 ul to a clean transparent PCR plate labeled “Plate1 PCR2 MIDPlate1 MIDPlate2

qPCR selection from both PCR2 plates and the pool

• Make 1:1000 dilutions of column 3 from the PCR2 plates by adding 1 ul to 999 ul TE in a deep well plate:

• Add 16 ul of Illumina Library Quantification MasterMix to each well:

• Add 4 ul of template, pool, or standards to each well:

• Run under standard conditions

Results:

Average results have been displayed below:

Brew_20_Nov_Col1_16S: 0.105 nanomoles

Brew_20_Nov_Col2_16S: 1.36 nanomoles

Brew_20_Nov_Col1_ITS: 3.88 nanomoles

Brew_20_Nov_Col2_ITS: 3.72 nanomoles

Brew_20_Nov_Col6_16S: 0.601 nanomoles

Brew_20_Nov_Col7_16S: 0.08 nanomoles

Brew_20_Nov_Col6_ITS: 5.06 nanomoles

Brew_20_Nov_Col7_ITS: 3.57 nanomoles

A full report of the results can be viewed below:

qPCR 16S and ITS Pool:

• Add 20uL from each well of the 16S plate into a 2mL sample tube and label “Brew 16S Pool”.

• Add 2uL from each well of the ITS plate into a 1.5mL sample tube and label “Brew ITS Pool”.

• Make 1:1000 dilutions of both the ITS and 16S pools.

• Add 16 ul of Illumina Library Quantification MasterMix to each well:

• Add 4 ul of template, pool, or standards to each well:

• Run under standard conditions

Results:

16S Pool: 0.48 nM

ITS Pool: 3.61 nM

Sequencing:

• 0.2 nM 16S Pool: 42.2 ul 16S Pool + 57.8 ul “10 mM Tris 8.5”

• 0.2 nM ITS Pool: 5.6 ul ITS Pool + 94.4 ul “10 mM Tris 8.5”

• 0.1 nM Full Pool: 50 ul from both 0.2 nM Pools

• Add 50 ul 0.1 nM Pool to 40 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX

• Remove iSeq100 Cartridge v2 from large freezer #3 36 hours to 1 week before sequencing and place in refrigerator 5 with room around it for full airflow

• Remove iSeq 100 i1 Flow Cell from refrigerator 5’s crisper drawer and open white foil pack and allow to equilibrate to RT for 10-15 minutes.

• Open “iSeq 100 i1 Reagent Cartridge v2”. Turn on iSeq100, sbsuser password: “GenomeTechnologies”.

• Click on “Sequence”. Watch Video. Do what video tells you to do. Follow on screen instructions until run starts.

• Results located: Data/SequencingRuns/”folder with applicable date”/Alignment_1/Fastq/*.fastq.gz