Ampliseq library low yield

[Sunny88]

Hello everyone,

I'm new in this forum and just joined it today. I noticed that this forum is awesome and full of experts.

I started to prepare ampliseq library recently, my amplicons are about 400bp. I run 30 different samples every time. For some reasons, some samples have high library yield, some have low yield. It is not because of samples DNA since I usually get high yield for some of these low yield samples when I repeat them. At the beginning, I thought I overdried the AMPure beads. Then, I changed to dry my beads 5 mins as the protocol suggests for the first purification after ligation. I dry 2-5 mins for the post library amplification. I still got similar results with some library prep has higher yield, some has very low yield. I wonder if anyone has the sample problem and if anyone knows the reasons.

Thank you all very much for your reply in advance.

[Sunny88]

Hello everyone,

I'm new in this forum and just joined it today. I noticed that this forum is awesome and full of experts.

I started to prepare ampliseq library recently, my amplicons are about 400bp. I run 30 different samples every time. For some reasons, some samples have high library yield, some have low yield. It is not because of samples DNA since I usually get high yield for some of these low yield samples when I repeat them. At the beginning, I thought I overdried the AMPure beads. Then, I changed to dry my beads 5 mins as the protocol suggests for the first purification after ligation. I dry 2-5 mins for the post library amplification. I still got similar results with some library prep has higher yield, some has very low yield. I wonder if anyone has the sample problem and if anyone knows the reasons.

Thank you all very much for your reply in advance.

[GA-JJ]

We have seen the same problem when 24 samples were prepared at the same time. We changed to do 16 samples only, the results are better, almost no failure anymore.

The reason for low yield lib, I guess, some well may not dry enough, any carryover may affect PCR amplification or cause amplicon lose.

Two things to share: 1.The lib concentration as low as 5nm on Q-PCR, may still give you good seq data; 2. Reduce blood/BMR input for DNA extraction if WBC counts super high , so you may get better quality of DNA to start with, then your lib may have good yield.

[Sunny88]

We have seen the same problem when 24 samples were prepared at the same time. We changed to do 16 samples only, the results are better, almost no failure anymore.

The reason for low yield lib, I guess, some well may not dry enough, any carryover may affect PCR amplification or cause amplicon lose.

Two things to share: 1.The lib concentration as low as 5nm on Q-PCR, may still give you good seq data; 2. Reduce blood/BMR input for DNA extraction if WBC counts super high , so you may get better quality of DNA to start with, then your lib may have good yield.

Thank you very much GA-JJ for sharing. I dry the beads for 5 min. I had problems when I dried beads longer (when I saw cracks). I still had problems for some samples with low concentrations when I did 18 samples in a batch. Not sure why. Thanks again for your reply.