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02-04-2022 Illumina agreed there was likely a consumables problem. They are sending a replacement cartridge next week.

This r-script was used to transform this LIMS report to this demux key.

iSeq Global Metrics

%Q30 Read 1	78.8
%Q30 Read 2	75.2
%Clusters PF	39
%Occupancy	90.2

%Clusters Passing Filter is concerning. We will reach out to Illumina.

Sample Processing

Plate: RMJan22

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)

ul/rxn	Reagent	# of rxns	ul needed
3	5X Kapa HiFi Buffer	80	330
0.45	10M dNTPs	80	50
0.3	Kapa HiFi HotStart DNA Pol	80	33
7.25	HPLC H2O	80	797
11	Total Volume	80	1210

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).

Plate	16S MID
RMJan22	16S0A4
RMJan22	16S0B4

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

Run on Thermocycler Program GSAF36:

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 (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

Pool 2uL from each well into a single pool. Make 1:10000 dilution of RMJan22 Pool and run in triplicate on qPCR.
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	50	500
2 ul	Primer Premix (10X)	50	100
4 ul	Ultra Pure Water	50	200
16 ul	Total Volume	50	800

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

	10	11	12
A	NTC	NTC	NTC
B	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	2 pM Std	2 pM Std	2 pM Std
G	20 pM Std	20 pM Std	20 pM Std
H

Results:

Average results for the following plates:

Full result report can be viewed below:

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.

100/Results = ul of Pool to Add

100/4.98 = 20uL of Pool to Add

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

100- 20 = 80uL 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:

Statistic	Percentage
%Q30 Read 1
%Q30 Read 2
%Clusters PF
%Occupancy

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