micro iSeq100 Pilot1

Sequenced 11-29-2020, data uploaded 11-30-2020

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:

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Phusion HF Buffer

450

1350

0.45

10M dNTPs

450

203

0.3

Kapa HiFi HotStart DNA Pol

450

135

3.25

HPLC H2O

450

1462

7

Total Volume

450

3150

  • 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:

16S0G6

16S0H6

ITS0E1

ITS0F1

16S0G6

16S0H6

ITS0E1

ITS0F1

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

Temp C

Cycles

Time

Temp C

Cycles

Time

95*

1X

3:00

98

15X

0:30

62

15X

0:30

72

15X

0:30

72

1X

5:00

4

1X

0:00

PCR2 Mastermix Preparation:

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Phusion HF Buffer

230

690

0.45

10M dNTPs

230

104

0.3

Kapa HiFi HotStart DNA Pol

230

69

0.5 ul

5 uM F and R FlowCell Primers

230

115

0.75

HPLC H2O

230

172

5

Total Volume

230

1150

  • 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

Temp C

Cycles

Time

Temp C

Cycles

Time

95*

1X

3:00

98

20X

0:30

55*

20X

0:30

72

20X

0:30

72

1X

5:00

4

1X

0:00


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:

 

1

2

3

4

5

6

7

8

9

10

11

12

 

1

2

3

4

5

6

7

8

9

10

11

12

A

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

NTC

NTC

NTC

B

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

2 pM Std

2 pM Std

2 pM Std

G

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

20 pM Std

20 pM Std

20 pM Std

H

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

 

 

 

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

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

10 ul

KAPA SYBR FAST qPCR MM (2X)

100

1000

2 ul

Primer Premix (10X)

100

200

4 ul

Ultra Pure Water

100

400

16 ul

Total Volume

100

1600

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

 

1

2

3

4

5

6

7

8

9

10

11

12

 

1

2

3

4

5

6

7

8

9

10

11

12

A

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

NTC

NTC

NTC

B

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

C

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.002 pM Std

0.002 pM Std

0.002 pM Std

D

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.02 pM Std

0.02 pM Std

0.02 pM Std

E

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

0.2 pM Std

0.2 pM Std

0.2 pM Std

F

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

2 pM Std

2 pM Std

2 pM Std

G

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

20 pM Std

20 pM Std

20 pM Std

H

Brew_20_Nov_Col1_16S

Brew_20_Nov_Col2_16S

Brew_20_Nov_Col1_ITS

Brew_20_Nov_Col2_ITS

Brew_20_Nov_Col6_16S

Brew_20_Nov_Col7_16S

Brew_20_Nov_Col6_ITS

Brew_20_Nov_Col7_ITS

 

 

 

 

  • 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:

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

10 ul

KAPA SYBR FAST qPCR MM (2X)

30

300

2 ul

Primer Premix (10X)

30

60

4 ul

Ultra Pure Water

30

120

16 ul

Total Volume

30

480

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

 

1

2

3

4

5

6

7

8

9

10

11

12

 

1

2

3

4

5

6

7

8

9

10

11

12

A

NTC

NTC

NTC

Brew_16S_Pool

 

 

 

 

 

 

 

 

B

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

Brew_16S_Pool

 

 

 

 

 

 

 

 

C

0.002 pM Std

0.002 pM Std

0.002 pM Std

Brew_16S_Pool

 

 

 

 

 

 

 

 

D

0.02 pM Std

0.02 pM Std

0.02 pM Std

 

 

 

 

 

 

 

 

 

E

0.2 pM Std

0.2 pM Std

0.2 pM Std

 

 

 

 

 

 

 

 

 

F

2 pM Std

2 pM Std

2 pM Std

 

 

 

 

 

 

 

 

 

G

20 pM Std

20 pM Std

20 pM Std

 

 

 

 

 

 

 

 

 

H

Brew_ITS_Pool

Brew_ITS_Pool

Brew_ITS_Pool

 

 

 

 

 

 

 

 

 

  • 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