qPCR of pools and phiX

qPCR

Make 1:1000 dilutions of all samples using 2 ml tubes
Add 16 ul of Illumina Library Quantification MasterMix to each well:

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	40	400
2 ul	Primer Premix (10X)	40	80
4 ul	Ultra Pure Water	40	160
16 ul	Total Volume	40	640

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

	1	2	3	4	5	6	7	8	9	10	11	12

	1	2	3	10	11	12
A	LRII.5a	LRII.5a	LRII.5a	NTC	NTC	NTC
B	LRII.5b	LRII.5b	LRII.5b	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std
C	locad2a	locad2a	locad2a	0.002 pM Std	0.002 pM Std	0.002 pM Std
D	locad2b	locad2b	locad2b	0.02 pM Std	0.02 pM Std	0.02 pM Std
E	0.05pM PhiX	0.05pM PhiX	0.05pM PhiX	0.2 pM Std	0.2 pM Std	0.2 pM Std
F	50pM PhiX	50pM PhiX	50pM PhiX	2 pM Std	2 pM Std	2 pM Std
G				20 pM Std	20 pM Std	20 pM Std
H

Results:

I did 2 separate 1:1000 dilutions (a and b) of the pools. I also did one 1:1000 dilution and one original of our 50 pM PhiX. The results seems to corroborate the ratios I saw in the analysis.

03-04-22 Results:

Sample Name	Detector	Task	Ct	StdDev Ct	Qty	Mean Qty

Sample Name	Detector	Task	Ct	StdDev Ct	Qty	Mean Qty
LRII.5a	Standards	Unknown	7.38	0.049	66.31	68.28	2.326
LRII.5a	Standards	Unknown	7.35	0.049	67.68	68.28	2.326
LRII.5a	Standards	Unknown	7.28	0.049	70.85	68.28	2.326
LRII.5b	Standards	Unknown	7.26	0.080	71.85	76.53	4.132
LRII.5b	Standards	Unknown	7.14	0.080	78.08	76.53	4.132
LRII.5b	Standards	Unknown	7.11	0.080	79.67	76.53	4.132
PhiX0.05	Standards	Unknown	17.56	0.127	6.11E-02	6.45E-02	5.76E-03
PhiX0.05	Standards	Unknown	17.56	0.127	6.12E-02	6.45E-02	5.76E-03
PhiX0.05	Standards	Unknown	17.34	0.127	7.11E-02	6.45E-02	5.76E-03
PhiX50	Standards	Unknown	7.08	0.084	81.04	84.63	4.970
PhiX50	Standards	Unknown	7.06	0.084	82.55	84.63	4.970
PhiX50	Standards	Unknown	6.93	0.084	90.30	84.63	4.970
RMJan22-IIa	Standards	Unknown	8.25	0.120	36.36	37.71	3.166
RMJan22-IIa	Standards	Unknown	8.29	0.120	35.45	37.71	3.166
RMJan22-IIa	Standards	Unknown	8.07	0.120	41.33	37.71	3.166
RMJan22-IIb	Standards	Unknown	8.21	0.118	37.41	41.17	3.259
RMJan22-IIb	Standards	Unknown	8.00	0.118	43.23	41.17	3.259
RMJan22-IIb	Standards	Unknown	8.01	0.118	42.87	41.17	3.259

This averages out to:

Sample	Avg Qty	Std Deviation

Sample	Avg Qty	Std Deviation
RMJan22-II	39.44	3.441
LRII.5	72.41	5.425
PhiX 50 pM	84.63	4.970
PhiX 0.05 pM	6.45e-002	5.76e-003
PhiX avg original	74.3

2-25-22 Results:

Sample Name	Detector	Task	Ct	StdDev Ct	Qty	Mean Qty	StdDev Qty

Sample Name	Detector	Task	Ct	StdDev Ct	Qty	Mean Qty	StdDev Qty
LRII.5_Red_Pool	Unknown	Unknown	9.31	0.605	16.65	12.97	5.204
LRII.5_Red_Pool	Unknown	Unknown	10.17	0.605	9.29	12.97	5.204
LRII.5_Red_Pool	Unknown	Unknown	5.7	2.734	196.45	106	74.13
RM_JAN22_II_Pool	Unknown	Unknown	8.14	0.220	36.98	32.25	4.786
RM_JAN22_II_Pool	Unknown	Unknown	8.34	0.220	32.36	32.25	4.786
RM_JAN22_II_Pool	Unknown	Unknown	8.58	0.220	27.41	32.25	4.786

There was a clear outlier in the LRII.5 pool trio to which we should have paid more attention. The difference from previous values appears to explain the altered ratios we saw.

RM_JAN22_II: 2*(39.44/32.25)=2.45

LRII.5: 72.41/12.97=5.58

calculated RM/LR: 0.44

reads RM/LR: 508910/1412494=0.36

expected RM/LR: 2

reads PhiX/Samples: 709274/1921404 = 0.37

Calculated PhiX/Samples: 0.28

expected PhiX/Samples: 0.167