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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

...

	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.

View file

name	NovaSeq2_PCR2redos.csv

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
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

View file

name	NovaSeq2Troubleshoot3.csv

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.

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