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Low Reads, Input DNA concentrations, and need for qPCR based product normalization

Low Reads, Input DNA concentrations, and need for qPCR based product normalization

Owned by Gregg Randolph
Last updated: Dec 08, 2021 by Linda van Diepen

Design complete Nov 11, 2021

Questions:

  1. How can we best accommodate low concentration input DNAs?

    1. Concentrate input DNAs?

    2. Concentrate/normalize low yield products?

    3. Both?

    4. Just put more volume of original DNA in?

  2. In the sequence yield, does input concentration, substrate, or some other factor correlate well with low read counts?

Investigations:

Bench(Question 1):

Experimental Sketch of crossed design:

  • Pull aliquots of some high read count and low read count samples.

  • For previously low yielding samples, repeat at typical template amount, and at 2x, and 4x volume added to the reaction (in column 2,3,4, and 6, 7, 8). 6x8 samples in low yield, 8 in high yield, 8 of mock community, 8 of blank (ISD only). 72 reactions with different templates.

  • Template layout is replicated/duplicated, with different barcodes for duplicates.

  • 1 set of 36 products will not be adjusted for pooling.

  • 1 set of 36 will be adjusted for pooling per qPCR results.

  • Use ISD at lower than typical concentrations for all for PCRs, as a positive internal control and recognizing that this will wreck reconstruction of absolute counts. Will add ISD to master mix.

  • Treat templates with previously high read counts in standard manner, except for lower concentration ISD.

Initial template setup:

  • Add 9 ul Ultrapure H2O to 4 columns of a plate

  • Transfer 1 ul of typical ISD/coligo columns to this plate, seal, vortex, spin down.

  • Add 30 ul of original templates to columns at other end of plate.

  • Add 2 ul of new ISD to template columns.

Start Reaction Plate Setup:

  • Add Ultrapure H2O to the reaction plates in the following pattern:

 

1

2

3

4

5

6

7

8

9

 

1

2

3

4

5

6

7

8

9

A

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

B

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

C

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

D

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

E

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

F

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

G

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

H

6ul

6ul

4ul

 

6ul

6ul

4ul

 

6ul

MasterMix (make for 16 plates in 50mL conical tube)

Dilute 30 ng/ul ISD to 0.001 pg/ul:

  • Add 33.3 ul ISD to 966.7 ul TE for 1 ng/ul

  • Add 1 ul of above to 999 ul TE for 1 pg/ul

  • Add 1 ul of above to 999 ul TE for 0.001 pg/ul or ~10% typical

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

200

600

0.45

10M dNTPs

200

90

0.3

Kapa HiFi HotStart DNA Pol

200

60

0.25

HPLC H2O

200

50

1

ISD

200

200

5

Total Volume

200

1000

  • Add 5 ul to each well of a hard shell, full skirt plate.

  • Add Templates following the pattern below:

Sample

Status

Plate

Location

Original Plate Name

Sample

Status

Plate

Location

Original Plate Name

SAG_S43P4R

Low Read

PA13

E11

AYAYEE_RHIZO_PLATE2

SAG_S10P4R

Low Read

PA12

H2

AYAYEE_RHIZO_PLATE1

SAG_S14P3R

Low Read

PA13

E1

AYAYEE_RHIZO_PLATE2

SAG191507

Low Read

PA1

B12

tube

SAG_S13P3R

Low Read

PA13

A1

AYAYEE_RHIZO_PLATE2

SAG_S3P2R

Low Read

PA12

B1

AYAYEE_RHIZO_PLATE1

SAG190267

Low Read

PA2

F5

tube

SAG190430

Low Read

PA1

B2

tube

SAG192203

Avg Read

PA2

F9

tube

SAG191508

Avg Read

PA1

A12

tube

SAG_S16P3R

Avg Read

PA12

G3

AYAYEE_RHIZO_PLATE1

SAG_S44P2R

Avg Read

PA12

F7

AYAYEE_RHIZO_PLATE1

 

1

2

3

4

5

6

7

8

9

 

1

2

3

4

5

6

7

8

9

A

pool 2 µL each

SAG192203

Tube 127

2ul SAG_S43P4R

PLT2: E11

4ul SAG_S43P4R

PLT2: E11

8ul SAG_S43P4R

PLT2: E11

mock community

2ul SAG_S13P3R

PLT2: A1

4ul SAG_S13P3R

PLT2: A1

8ul SAG_S13P3R

PLT2: A1

 blank with ISD only

B

pool 2 µL each

SAG191508

Tube 73

2ul SAG_S10P4R

PLT1: H2

4ul SAG_S10P4R

PLT1: H2

8ul SAG_S10P4R

PLT1: H2

mock community

2ul SAG_S3P2R

PLT1: B1

4ul SAG_S3P2R

PLT1: B1

8ul SAG_S3P2R

PLT1: B1

blank with ISD only

C

pool 2 µL each

SAG_S16P3R

PLT1: G3

2ul SAG_S14P3R

PLT2: E1

4ul SAG_S14P3R

PLT2: E1

8ul SAG_S14P3R

PLT2: E1

mock community

2ul SAG190267

Tube 259

4ul SAG190267

Tube 259

8ul SAG190267

Tube 259

  blank with ISD only

D

pool 2 µL each

SAG_S44P2R

PLT1: F7

2ul SAG191507

Tube 74

4ul SAG191507

Tube 74

8ul SAG191507

Tube 74

mock community

2ul SAG190430

Tube 10

4ul SAG190430

Tube 10

8ul SAG190430

Tube 10

  blank with ISD only

E

pool according to yield

SAG192203

Tube 127

2ul SAG_S43P4R

PLT2: E11

4ul SAG_S43P4R

PLT2: E11

8ul SAG_S43P4R

PLT2: E11

mock community

2ul SAG_S13P3R

PLT2: A1

4ul SAG_S13P3R

PLT2: A1

8ul SAG_S13P3R

PLT2: A1

 blank with ISD only

F

pool according to yield

SAG191508

Tube 73

2ul SAG_S10P4R

PLT1: H2

4ul SAG_S10P4R

PLT1: H2

8ul SAG_S10P4R

PLT1: H2

mock community

2ul SAG_S3P2R

PLT1: B1

4ul SAG_S3P2R

PLT1: B1

8ul SAG_S3P2R

PLT1: B1

blank with ISD only

G

pool according to yield

SAG_S16P3R

PLT1: G3

2ul SAG_S14P3R

PLT2: E1

4ul SAG_S14P3R

PLT2: E1

8ul SAG_S14P3R.

PLT2: E1

mock community

2ul SAG190267

Tube 259

4ul SAG190267

Tube 259

8ul SAG190267

Tube 259

  blank with ISD only

H

pool according to yield

SAG_S44P2R

PLT1: F7

2ul SAG191507

Tube 74

4ul SAG191507

Tube 74

8ul SAG191507

Tube 74

mock community

2ul SAG190430

Tube 10

4ul SAG190430

Tube 10

8ul SAG190430

Tube 10

  blank with ISD only

  • Add 2 ul 1-step primers:

  • Seal with bubble seals. Vortex briefly. Spin down.

  • Run on Thermocycler Program GSAF36:

Temp C

Cycles

Time

Temp C

Cycles

Time

95*

1X

3:00*

98

36X

0:30

62

36X

0:30

72

36X

0:30

72

1X

5:00

4

1X

0:00

MagBead Cleanup:

Some plates were cleaned up using the Nimbus platform protocol “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 replicate

  • 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 (Plate name_PCR_MIDs)

qPCR all products:

  • Make 1:1000 dilutions of all samples from the PCR 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

SAG192203

Tube 127

2ul SAG_S43P4R

PLT2: E11

4ul SAG_S43P4R

PLT2: E11

8ul SAG_S43P4R

PLT2: E11

mock community

2ul SAG_S13P3R

PLT2: A1

4ul SAG_S13P3R

PLT2: A1

8ul SAG_S13P3R

PLT2: A1

 blank with ISD only

 

 

NTC

B

SAG191508

Tube 73

2ul SAG_S10P4R

PLT1: H2

4ul SAG_S10P4R

PLT1: H2

8ul SAG_S10P4R

PLT1: H2

mock community

2ul SAG_S3P2R

PLT1: B1

4ul SAG_S3P2R

PLT1: B1

8ul SAG_S3P2R

PLT1: B1

blank with ISD only

 

04.0002 pM Std

0.0002 pM Std

C

SAG_S16P3R

PLT1: G3

2ul SAG_S14P3R

PLT2: E1

4ul SAG_S14P3R

PLT2: E1

8ul SAG_S14P3R

PLT2: E1

mock community

2ul SAG190267

Tube 259

4ul SAG190267

Tube 259

8ul SAG190267

Tube 259

  blank with ISD only

 

0.002 pM Std

0.002 pM Std

D

SAG_S44P2R

PLT1: F7

2ul SAG191507

Tube 74

4ul SAG191507

Tube 74

8ul SAG191507

Tube 74

mock community

2ul SAG190430

Tube 10

4ul SAG190430

Tube 10

8ul SAG190430

Tube 10

  blank with ISD only

 

0.02 pM Std

0.02 pM Std

E

SAG192203

Tube 127

2ul SAG_S43P4R

PLT2: E11

4ul SAG_S43P4R

PLT2: E11

8ul SAG_S43P4R

PLT2: E11

mock community

2ul SAG_S13P3R

PLT2: A1

4ul SAG_S13P3R

PLT2: A1

8ul SAG_S13P3R

PLT2: A1