Rob McMinn iSeq Run (loc_ad1)

Plate: 5RM1 (due to low extraction yields, this plate was completed on the iSeq)

NOTE: Plate sequenced for 16S only but ITS was prepped up until MagBead. No MagBead clean up was completed on ITS but is available if client would like to sequence for ITS in the future.

MasterMix (make for 4 plates in 5 mL tube)

• Add 11 ul to each well of a hard shell, full skirt plate. Seal with bubble strips and store in refrigerator until needed label “NovaSeq4 PCR”. (If doing manually, one needs 2 ul of template and 2 ul of the primers).

*ITS plates PCR’d but did not continue to MagBead clean up.

Run on Thermocycler Program GSAF36:

MagBead Cleanup (16S Only):

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

• Make 1:1000 dilutions of column 1, 4, and 8 from the PCR plates by adding 1 ul to 999 ul TE in a deep well plate:

• Pool 2uL from each well into a single pool. Make 1:1000 dilution of 5RM1 Pool and run in duplicate on qPCR.

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

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

Results:

Average results for the following plates:

5RM1_16S_Col1: 0.489 nanomoles

5RM1_16S_Col4: 0.882 nanomoles

5RM1_16S_Col8: 1.29 nanomoles

5RM1_16S_Pool: 2.88 nanomoles

Full result report can be viewed below:

Open

iSeq Run:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

• 1000/Results = ul of Pool to Add

1000/2.88 = 347uL of Pool to Add

• 1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 347 = 653uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 50 pM:

• Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX

• 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

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

The sequencing run showed poor global statistics:

A double check of the 1nM pool dilution created for sequencing revealed it was actually 4.98 nM. We will repeat sequencing run.

iSeq Run 2:

Dilute to 1 nM based off qPCR results. qPCR results are in pM, but 1:1000 dilution used. The results are effectively in nM for pool.

• 1000/Results = ul of Pool to Add

1000/4.98 = 200uL of Pool to Add

• 1000 - uL of Pool to Add = ul of “10 mM Tris 8.5” to Add

1000- 347 = 800 uL of 10mM Tris 8.5

Dilute 1 nM full pool to loading concentration of 50 pM:

• Add 5 ul 1 nM Pool to 85 ul “10 mM Tris 8.5” and 10 ul 50 pM PhiX

• 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

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