Even Read Distribution in Multiplexed Runs

[nangel]

There have been a few old posts but nothing recent.

We are interested to know how even people are managing to get reads for each sample pooled at equimolar amounts in a run.

Our team performs library preparations with standard Illumina DNA prep libraries and supplied 384 UDI indexes.

Quantification via flourometric assay, and gel image of each library to get the average size and convert to an accurate nM measure for each library.

We have tried qPCR quantification also.

We monitor for small fragments and primer dimers etc.

Our question is how much variance between sample reads is generally accepted (potentially due to indexes used?- there seems to be some supporting literature on the Illumina website that suggests this can be a factor).

Are other groups seeing something like +/- 3 million reads when attempting to pool evenly at 10 million reads per sample for example.

We would look to further refine but are unsure how tightly this metric can be controlled?

Thanks for anyone else's experience, insight, tips or tricks!

[Ben3]

Hello nangel I was doing this not that long again. I was using fluorometric assays for concentration and then used automated electrophoresis for the average size and to monitor for dimers.

I tried using qPCR many times, but I felt like it was even more variable. I knew groups that only used qPCR but I struggled to get even measurements every time.

But to answer your question. I was seeing some variance like this but I was probably running lower plex libraries than you and getting many more reads per sample.

I feel like this will always be hard to control because small variations in pipetting and quantification will throw off the number of reads.

A few things I started to do to help were dilute down samples that were of higher concentration. Then I would check the concentration again before pooling. Sometimes I would check the concentration twice for each sample and take the average to ensure I got as close as I could to the true concentration. I also upgraded my pipettes and only used a P10 and P2 for most of my dilutions.

These are all tedious suggestions and not perfect, but I feel like they helped keep the concentrations more consistent than they were. Hopefully they help and good luck!