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Gregg Randolph Linda van Diepen Alex Buerkle

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1. Pull aliquots of some high read count and low read count samples. (Pending new low read samples).

2. Quantify all samples not including blanks or mock community.

3. 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. See template below for clarification.

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

5. 1 set of 36 products will not be adjusted for pooling.

6. 1 set of 36 will be adjusted for pooling per qPCR results.

Start Reaction Plate Setup:

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Red samples (bottom half) will either just be pooled per qPCR numbers or concentrated via SpeedVac, reconstituted to a higher concentration, and pooled by qPCR results. (Pending method instructions).

“ul for pooling” was calculated by multiplying the lowest molarity (3.15) times the maximum V available (35 ul) to get ~110. This result was then divided by each sample’s molarity to obtain the ul needed for equimolar pooling rounded to one decimal place.

This will need to be done by hand, but if we pursue this I will tweak the Nimbus normalization program to do this. For a NovaSeq run, I would pick a higher minimum molarity. While this pool should be above the NovaSeq minimum molarity needed of 1 nM at 3-4 nM, being higher than its 3.15 nM minimal constituent, I would prefer a larger margin of safety like 5 nM. For an iSeq run, we dilute down to 50 pM, so it is not an issue.

qPCR blue/red pools:

• Make 1:1000 dilutions of blue and red pools by adding 1 ul to 999 ul TE in a 1.5mL tubes.

• Run each pool in triplicate.

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

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Average Results:

LR2_Blue_Pool: 53.65 nanomoles

LR2_Red_Pool: 35.33 nanomoles

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.

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• 1000/Results = ul of Pool to Add

1000/____= ____ 53.7 = 18.6 uL of Pool to Add

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

1000- ____ = ____ 18.6 = 981.4 uL of 10mM Tris 8.5

Red Pool

• 1000/Results = ul of Pool to Add

1000/___ = ___ 35.3 = 28.3 uL of Pool to Add

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

1000- ___= ___ 28.3= 971.7 uL of 10mM Tris 8.5

Combine Blue and Red pools in equal parts once pools are at 1nM

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Use R to associate NS2 input concentrations with sample names, read counts, and % ISD reads

Repeat qPCR post calibration:

qPCR MMX

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

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

Results 02/08/2022:

Results for duplicate qPCR run on 02/09/2022:

Results look the same from the 02/08 run.