2 -step Library preparation protocol for 16s or ITS

Aliquot 30 ul of full concentration DNA extract

Add 6 ul of control oligo pool to environmental DNA aliquot (16S and ITS coligo 0.01 pg/ul each AND 16S SG_GR and ITS SG_GR 0.03 pg/ul each

16S Coligo: 5'-GTGCCAGCAGCCGCGGTAA AACAACAACAACC ATTAGATACCCTAGTAGTCC-3'

Coligo Structure: 5'-LocusPrimer Barcode InverseLocusPrimerR -3'

ITS Coligo: 5'-CTTGGTCATTTAGAGGAAGTAAT AACAACAACAACC CGTAGCTACTTCTTGCGTCG-3'

https://microcollaborative.atlassian.net/wiki/spaces/MICLAB/pages/837910529

Sample tracking in microbiome community profiling assays using synthetic 16S rRNA gene spike-in controls

Synthgene Structure: 5'-LocusPrimer Nonsense InverseLocusPrimer-3'

ITS Synthgene:

CTTGGTCATTTAGAGGAAGTAATGCCACAGATACGTACCGCTCATAACGCGAACCGAAGCGCAGTAGAAGTACTCCGTATCCTACCTCGGTCGTGGTTTAGGCTATCGACATCTTGCATGGGCTTCCCTAGTGAACTCTTGGGATGTGCATCGATGAAGAACGCAGC

Alignment of primers and synthetic amplicon control molecules (synthgenes): Synthgene_detective.pdf (also on OneDrive).

16S Synthgene:

GTGCCAGCAGCCGCGGTAAGCCACAGATACGTACCGCTCATAACGCGAACCGAAGCGCAGTAGAAGTACTCCGTATCCTACCTCGGTCGTGGTTTAGGCTATCGACATCTTGCATGGGCTTCCCTAGTGAACTCTTGGGATGTATTAGATACCCTAGTAGTCC

https://microcollaborative.atlassian.net/browse/FS2017DNA-13

Tourlousse, D. M., Yoshiike, S., Ohashi, A., Matsukura, S., Noda, N., & Sekiguchi, Y. (2017). Synthetic spike-in standards for high-throughput 16S rRNA gene amplicon sequencing

 

Normalize eDNA to 10 ng/ul

 

Add 7 ul Master mix to each well of a new plate:

ul/rxn

Reagent

ul/rxn

Reagent

3

5X KAPA HiFi HotStart PCR Buffer

0.45

10M dNTPs

0.3

Kapa HiFi HotStart DNA Pol

3.25

HPLC H2O

7

Total Volume

Add 6 ul appropriate 0.25 uM paired primers Molecular IDentifier (MID) Plate:______________

16S Primer Base

  • 515F (5’-GTGYCAGCMGCCGC GGTAA-3’) (Parada et al. 2016)

  •  806R (5’-GGACTACNVGGGTWTCTAAT-3’) (Apprill et al. 2015)

ITS Primer Base

Primer structure

  • 5'-PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeGTGYCAGCMGCCGCGGTAA-3'

  • 5'-PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeGGACTACHVGGGTWTCTAAT-3'

  • 5'-PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeCTTGGTCATTTAGAGGAAGTAA-3'

  • 5'-PARTIAL_ILLUMINA_FLOWCELL_SEQbarcodeGCTGCGTTCTTCATCGATGC-3'

Actual Sequences: https://microcollaborative.atlassian.net/wiki/pages/createpage.action?spaceKey=MICLAB&title=Information%20on%20oligos&linkCreation=true&fromPageId=351141889

Each 515F/806R and ITS1F/ITS2 were arrayed to create all 9216 possible MID pairings. The first MID array was a stamp of both plates (A1 in A1 for both plates) and was labeled 16S0A1 or ITS0A1. The second plate was a stamp of the reverse plate but the forward plate was rotated so that B1 went in A1, C1 in B1, D1 in C1, . . . , H12 in G12 and A1 in H12. This second plate was labeled 16S0B1 or ITS0B1 referring to the original position of the forward oligo plate MID that was located in A1 of the arrayed plate. This schema was continued around until 16S0H12 and ITS0H12 for 96 total MID plates for each locus.

Add 2 ul of modified eDNA aliquot to each well

Run on Thermocycler Program GSAF1:

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

Pool duplicates together.

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

Pool Duplicate PCR reactions (Transfer 15 ul of one replicate to the other)

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 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.

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

Add 5 ul FlowCell MasterMix to new plate:

ul/rxn

Reagent

# of rxns

ul needed

ul/rxn

Reagent

# of rxns

ul needed

3

5X Kapa HiFi Buffer

60

210

0.45

10M dNTPs

60

31.5

0.3

Kapa HiFi HotStart DNA Pol

60

21

0.5 ul

10 uM F and R FlowCell Primers

60

35

0.75

HPLC H2O

60

52.5

5

Total Volume

60

350

FlowCell Primers

  • AATGATACGGCGACCACCGAGATCTACACTCGTCGGCAGCGTC

  • CAAGCAGAAGACGGCATACGAGATGTCTCGTGGGCTCGG

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

Run on Thermocycler Program GSAF2:

Temp C

Cycles

Time

Temp C

Cycles

Time

95*

1X

3:00*

98

19X

0:30

55*

19X

0:30

72

19X

0:30

72

1X

5:00

4

1X

0:00

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 30 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 40 ul TE; 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 PCR plate.

Use Synergy HTX’s Take 3 Trio to quantify DNA via absorbance.

Spot Check some PCR rxn’s on Agilent Bioanalyzer 2100 DNA 1000 chip

Normalize PCR’s via absorbance concentrations. @Gregg Randolph What concentration did you normalize to? Or has this not been finalized yet?

Pool normalized PCRs

Check Pool’s molar concentration via qPCR:

Dilute Pool 1:1000

Include NTC and Standards (20 pm, 2 pm, 0.2, 0.02pm, 0.002pm, 0.0002pm)

ul/rxn

Reagent

ul/rxn

Reagent

10 ul

KAPA SYBR FAST qPCR MM (2X)

2 ul

Primer Premix (10X)

4 ul

Ultra Pure Water

16 ul

Total Volume

qPCR

Temp C

Cycles

Time

Temp C

Cycles

Time

94

1X

0:01

95

1X

5:00

95

32X

0:30

60

1:00

If necessary, concentrate via Vacuum Concentrator (SpeedVac DNA130)

Complete Libraries are sent to the Genomic Sequencing and Analysis Facility at the University of Texas to be run on their NovaSeq 6000 using paired end 2x250 chemistry. It will be treated like a low diversity library with a likely 10% PhiX spike in.

Small format libraries may be run on our iSeq100 with a 10% PhiX spike-in after dilution to 60 pM.