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Hi, my lab has been using MiSeq for quite sometime now.
However, out of all our runs this has been the worst result yet.
Cluster Density: 367K/mm2
Clusters Passing Filter: 48.3%
Estimated Yield: 2941.4 MB
Q30: 17.8%
I was hoping to get everyone's help on this topic.
I do know that the normalization part is especially important.
We tried to make our sample 0.8 ng/uL (original concentration) in our qubit, which is about 2nM.
However, after pooling and measuring the qubit we got 0.35 ng/uL.
We still decided to run with it because we couldn't afford to normalize 384 samples all again.
Our PhiX is at 30% with 2nM, and the 0.2N NaOH we used was pH of 13.33. (NaOH was made 5 hours before used)
We followed the procedure as follow.
a. pooled library + NaOH (5ul each)
b. PhiX + NaOH (5ul each)
c. 5 minutes of denaturation
d. Mix 10ul of sample a,b with 990ul of HT1.
e. Then get 700 ul of pooled library + NaOH + HT1 with 300 ul of PhiX + NaOH + HT1.
f. Lastly we set the pM at 8pM
Then we ran it and got a low cluster density.
We plan to try saving this sample. We were thinking of doing this in either ways. Which is preferable
1. Rerun the sample with the same pooled library at 16pM or even higher.
2. Normalize the samples all over again, and this time match it to 0.8 ng/uL which will equal to 2nM. Then run it again with 8pM.
I was hoping to get some answers also to these other questions since Illumina does not seem to answer all our answers honestly or they really just don't know.
1. Do you think the problem has to do with the library quantification itself? Is the concentration of the sample crucial?
2. Why is it that when our densitiy is high, we also don't get good results?
3. Are there ways to choose the pM of the samples without running the sample and rerun it again? I feel like using another catridge is such a waste of money.
Thank you in advance. I really hope to get some help from you guys.
However, out of all our runs this has been the worst result yet.
Cluster Density: 367K/mm2
Clusters Passing Filter: 48.3%
Estimated Yield: 2941.4 MB
Q30: 17.8%
I was hoping to get everyone's help on this topic.
I do know that the normalization part is especially important.
We tried to make our sample 0.8 ng/uL (original concentration) in our qubit, which is about 2nM.
However, after pooling and measuring the qubit we got 0.35 ng/uL.
We still decided to run with it because we couldn't afford to normalize 384 samples all again.
Our PhiX is at 30% with 2nM, and the 0.2N NaOH we used was pH of 13.33. (NaOH was made 5 hours before used)
We followed the procedure as follow.
a. pooled library + NaOH (5ul each)
b. PhiX + NaOH (5ul each)
c. 5 minutes of denaturation
d. Mix 10ul of sample a,b with 990ul of HT1.
e. Then get 700 ul of pooled library + NaOH + HT1 with 300 ul of PhiX + NaOH + HT1.
f. Lastly we set the pM at 8pM
Then we ran it and got a low cluster density.
We plan to try saving this sample. We were thinking of doing this in either ways. Which is preferable
1. Rerun the sample with the same pooled library at 16pM or even higher.
2. Normalize the samples all over again, and this time match it to 0.8 ng/uL which will equal to 2nM. Then run it again with 8pM.
I was hoping to get some answers also to these other questions since Illumina does not seem to answer all our answers honestly or they really just don't know.
1. Do you think the problem has to do with the library quantification itself? Is the concentration of the sample crucial?
2. Why is it that when our densitiy is high, we also don't get good results?
3. Are there ways to choose the pM of the samples without running the sample and rerun it again? I feel like using another catridge is such a waste of money.
Thank you in advance. I really hope to get some help from you guys.