Gregg Randolph Linda van Diepen Alex Buerkle
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Pull aliquots of some high read count and low read count samples. (Pending new low read samples).
Quantify all samples not including blanks or mock community.
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.
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.
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.
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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.
Results located: Data/SequencingRuns/”folder with applicable date”/Alignment_1/Fastq/*.fastq.gz
Upload data through Globus portal off external drive
Data wrangling(2):
Use R to associate NS2 input concentrations with sample names, read counts, and % ISD reads