NovaSeq2 Troubleshoot

The library pool was flagged by Psomagen as suspect. They could not get it to qPCR and the size was too small by approximately the extension accomplished via PCR2.

Psomagen QC

Enlarged Electropherogram from Psomagen

We initially tried qPCR on the full strength pool and a 1:1000 dilution.

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	24	300
2 ul	Primer Premix (10X)	24	60
4 ul	Ultra Pure Water	24	120
16 ul	Total Volume	24	480

Add 4 ul of templates

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

	1	2	3	4
A	NTC	NTC	NTC	1:1000 N2
B	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std	1:1000 N2
C	0.002 pM Std	0.002 pM Std	0.002 pM Std	1:1000 N2
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	NovaSeq2	NovaSeq2	NovaSeq2

N2=NovaSeq2

The results confirmed the low concentration reported. We did get amplification though.

The full strength gave a concentration of 6 pMoles/ul and the dilution gave a thousandth of that confirming each other.

We tried to concentrate the library, but after researching the requirements for the NovaSeq loading concentration realized that would not work.

We went back to some of the individual plates to investigate possible problems. We redid PCR2 of one of the first plates done and one of the last from the saved and frozen PCR1s and compared them with the original PCR1s via qPCR of 1:1000 dilutions.

ul/rxn	Reagent	# of rxns	ul needed

ul/rxn	Reagent	# of rxns	ul needed
10 ul	KAPA SYBR FAST qPCR MM (2X)	53	600
2 ul	Primer Premix (10X)	53	120
4 ul	Ultra Pure Water	53	240
16 ul	Total Volume	53	960

Add 4 ul template:

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

	1	2	3	4	5	6	7
A	NTC	NTC	NTC	PE1 A1	JC1 A1	PE1 A1 Take 2	JC1 A1 Take2
B	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std	PE1 B1	JC1 B1	PE1 B1 Take 2	JC1 B1 Take 2
C	0.002 pM Std	0.002 pM Std	0.002 pM Std	PE1 C1	JC1 C1	PE1 C1 Take 2	JC1 C1 Take 2
D	0.02 pM Std	0.02 pM Std	0.02 pM Std	PE1 D1	JC1 D1	PE1 D1 Take 2	JC1 D1 Take 2
E	0.2 pM Std	0.2 pM Std	0.2 pM Std	PE1 E1	JC1 E1	PE1 E1 Take 2	JC1 E1 Take 2
F	2 pM Std	2 pM Std	2 pM Std	PE1 F1	JC1 F1	PE1 F1 Take 2	JC1 F1 Take 2
G	20 pM Std	20 pM Std	20 pM Std	PE1 G1	JC1 G1	PE1 G1 Take 2	JC1 G1 Take 2
H				PE1 H1	JC1 H1	PE1 H1 Take 2	JC1 H1 Take 2

Well, there is certainly a difference between the initial PCR2 and the redone version. There is a 100 fold increase in the redone version.

We will repeat this one more time with the flow cell primers created anew from the 300 uM stock using one plate, PE1. We will also do a repeated PCR1 for this plate to include in the qPCR comparison.

1:1000 dilutions of final PCRs loaded.

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

	1	2	3	4	5	6	7
A	NTC	NTC	NTC	PE1 A1 Take 2	PE1 A1 Take 1	PE1 A1 Take 3	PE1 A1 Take 4
B	0.0002 pM Std	0.0002 pM Std	0.0002 pM Std	PE1 B1 Take 2	PE1 B1 Take 1	PE1 B1 Take 3	PE1 B1 Take 4
C	0.002 pM Std	0.002 pM Std	0.002 pM Std	PE1 C1 Take 2	PE1 C1 Take 1	PE1 C1 Take 3	PE1 C1 Take 4
D	0.02 pM Std	0.02 pM Std	0.02 pM Std	PE1 D1 Take 2	PE1 D1 Take 1	PE1 D1 Take 3	PE1 D1 Take 4
E	0.2 pM Std	0.2 pM Std	0.2 pM Std	PE1 E1 Take 2	PE1 E1 Take 1	PE1 E1 Take 3	PE1 E1 Take 4
F	2 pM Std	2 pM Std	2 pM Std	PE1 F1 Take 2	PE1 F1 Take 1	PE1 F1 Take 3	PE1 F1 Take 4
G	20 pM Std	20 pM Std	20 pM Std	PE1 G1 Take 2	PE1 G1 Take 1	PE1 G1 Take 3	PE1 G1 Take 4
H				PE1 H1 Take 2	PE1 H1 Take 1	PE1 H1 Take 3	PE1 H1 Take 4

Take 1: Original Prep

Take2: PCR2 redone from original PCR1 with original flow cell primer dilutions

Take3: PCR2 redone from original PCR1 with new dilutions of flow cell primers from original 300 uM stock

Take 4: Fresh full prep using new dilutions of flow cell primers and original normalized template plate

It looks like I did something wrong with my dilution of the flow cell primers. I should maybe do a test pcr before rolling with a new dilution. Both the completely redone pcrs and those redone from the original PCR1s gave good results within the expected range. Both, once adjusted for dilution were in the single to double digit nano molar range. I will proceed with redoing PCR2 from the saved PCR1s.