micro NovaSeq Run #3

Status (23 September): Library was submitted to Psomagen on 21 September and received on 22 September. Electropherogram QC returned 23 September. There is evidence of some high bp fragments. They are proceeding with sequencing (9-28-2020)

QC From Psomagen:

183bp is coligos. 322 is Synthgene. 458 is the sample products, with a skew towards 16S over ITS.

Sequencing Report from Psomagen:

Sample ID

Total Read Bases(bp)

Total Reads

GC(%)

AT(%)

Q20(%)

Q30(%)

NovaSeq3

3.82E+11

1.52E+09

63.62

36.38

90.87

82.85

  • Create MasterMix for a third of the reactions at a time:

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

1650

4950

0.45

10M dNTPs

1650

743

0.3

Kapa HiFi HotStart DNA Pol

1650

495

3.25

HPLC H2O

1650

5362

7

Total Volume

1650

11550

  • Add 7 ul to each well of 16 hard shell, full skirt plates, Seal with tape seals, rub with kimwipe across all wells twice, and then around the edge, store in refrigerator until needed labeled “PCR1”. (If doing manually, one needs 2 ul of template and 6 ul of the primers).

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

  • Open “TwoLociDuplicatePCR_Prep_BR” in the Nimbus Method Editor

  • Press the stop light icon to open Hamilton Run Control

  • Press the green triangle

  • Load 3 of the MID plates, the 4 PCR1 plates, and the “_Norm” plate (template) according to the prompts and 5 of the 50 ul conductive, sterile, filtered tips.

  • When prompted, swap the “_Norm” with the 4th MID plate.

  • 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

  • Store at 4C when cycler reaches below 40C unless immediately proceeding to next step

  •  

Cleanup of PCR 1 product:

  • This was done on the Nimbus platform using “AxyPrep MagBead PCR1 No MM”

    and manually. Probably 3/4 of the plates were cleaned manually.

  • Manual 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 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 30 ul H2O; 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 30 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

PCR 2

Create MasterMix2 for 1/3 of the reactions:

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Phusion HF Buffer

810

2430

0.45

10M dNTPs

810

364.5

0.3

Kapa HiFi HotStart DNA Pol

810

243

0.5 ul

5 uM F and R FlowCell Primers

810

405

0.75

HPLC H2O

810

607.5

5

Total Volume

810

4050

Add 5 ul master mix to all wells of 4 hard shell full skirted plates (the cheaper soft, skirted plates don’t seal very well regardless of caps used). I am using the new green plates Gregg got. Seal with tape seals and store in refrigerator if not using immediately. Add 10ul of template from PCR1.

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

Pooling:

Benchsmart 96 used to transfer 2 ul from each well of 24 plates to a

QA Checks:

Bioanalyzer:

Ran Yoon Yoon Plate by itself and the rest of the pool by itself on the bioanalyzer

 

 

qPCR:

We initially tried qPCR on the full strength pool and a 1:1000 dilution.

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

10 ul

KAPA SYBR FAST qPCR MM (2X)

24

300

2 ul

Primer Premix (10X)

24

60

4 ul

Ultra Pure Water

24

120

16 ul

Total Volume

24

480

Add 4 ul of 1:1000 templates and standards

 

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

NovaSeqYoon

 

 

 

 

 

 

 

 

B

0.0002 pM Std

0.0002 pM Std

0.0002 pM Std

NovaSeqYoon

 

 

 

 

 

 

 

 

C

0.002 pM Std

0.002 pM Std

0.002 pM Std

NovaSeqYoon

 

 

 

 

 

 

 

 

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

NovaSeq3

NovaSeq3

NovaSeq3

 

 

 

 

 

 

 

 

 

Both pools were 20-25 nMolar with Yoon being closer to 20 and the Full being closer to 25.