Low concentration and large size of Trusight One library

[Merav]

Hi!

I encountered two problems while preparing Illumina's Trusight One library using their guide. I'm following the protocol carefully (except from adjusting the protocol to tubes instead of plates) and started from a total of 50ng of high quality human genomic DNA.

1. I'm getting low concentration (about half of the lower threshold) of the enriched library. The enriched library is prepared on a pool of 12-plex pre-enriched libraries, and the pre-enriched libraries are in good concentration (60-90 ng/ul each). I already repeated twice and got exactly the same results.

2. The library size is big. In the first time the average size (as measured by BioAnalyzer) was 600 bp, and in the second in was 520 bp. I'm measuring the input as accurately as possible DNA in triplicates using Qubit.

Need your advices, ideas and anything you can think that can help me improve this.

Thanks!
Merav

[ymzhong]

The size of your library is good. It's about 500bp according to the instruction. I did Trusight One twice so far, one was 640pb, and the other one was 502bp.

What concentration (nM) did you get? What do you mean " half of the lower threshold"? The concentrations of my two library pools were also low, 1.90nM and 1.49nM. I need to figure out how to improve the final product.

[Merav]

Hi,
Our libraries concentration was ~ 24nM.
After a long time of trouble shooting according to Illumina tech support's suggestions, eventually, they said it was OK from the start…
We sequenced so far 2 Trusight ONE libraries and they were very good.

[ymzhong]

You library concentration was really good, much higher than mine. My pre-enriched libraries are in good concentrations as yours (~90 ng/ul).

I finished the sequencing of the pooled library (two samples) with a concentration of 1.90 nM and the results are good.

I contacted the illunima, but they do not have specific suggestions for improving the yield, just saying a concentration larger than 1.25nM is enough for sequencing.

I am glad yours worked very well!

[ymzhong]

Could you please help with my TruSight One?

I keep getting pooled libraries with very low concentrations (1-2nM in 30ul). Since I usually do 2 samples at one time, I use tube instead of plate for making libraries, and mix samples (with beads) by pipetting instead of shaking. What do you usually do and what type of magnetic stand do you use?

For my recent run last week, the concentration of final pooled libraries is 1.1nM (by real-time PCR), and I loaded a final concentration of 15pm (instead of 12.5pm) of library onto the MiSeq. The cluster density is about 600k/mm2, which is expected to be 1200-1400K/mm2, and the 20x coverage is only around 91%.

Thanks,
Yiming

[gabrieltw]

How did you load 15pM from 1.1 nM library ?

[ymzhong]

Combine 13.6ul of 1.1nM library and 13.6ul of 0.1N NaOH, mix well, incubate at room temp for 5 minutes, then add 965.3ul HT1 and 7.5ul of 20pM PhiX. Thanks.

[gabrieltw]

I usually try to keep my NaOH concentration less than 1 mM in loading library. I noticed that illumina is using 0.1 N on miniseq library dilution. I have never tried it with 0.1N, I use 0.2N from MiSeq protocol

[ymzhong]

The TrueSight One protocol for MiSeq uses 0.1N NaOH.

[ymzhong]

Hi, gabrieltw, do you have any suggestions to improve the final yield of pooled library? Thanks!

[gabrieltw]

How you do pool your library ?

If you use beads, just use less elution buffer to elute your library or just use SpeedVac to increase the concentration ?

[ymzhong]

Yes, I used beads. The final elution volume was about 30uL.

Do you use PCR tubes or plates for library production? The protocol is for plates, but I use tubes because I usually work with 2 samples every time.

Thanks.

[gabrieltw]

Yes, I used beads. The final elution volume was about 30uL.

Do you use PCR tubes or plates for library production? The protocol is for plates, but I use tubes because I usually work with 2 samples every time.

Thanks.

I work with both. Since you are using tubes, it's actually very easy to reduce the elution volume. I regularly elute in 10-15 uL using eppendorf tube

[ymzhong]

Thanks, I will try that.

What concentration do you usually get? Mine are 1-2nM, really low.

[Number 4]

Hi there,

What concentration of Trusight One DNA do you all load onto the Hiseq 2500? and what kind of yield do you yet? Trying to avoid a case of trial and error (£££!) so hoping to learn from other peoples experiences

Thanks!