Problem with the library DNA quantity and quality

[davalbuq]

Hi all,

It is totally new on the NGS world. Today, I started to prepare my first library preparation by using SureSelect Agilent technology. I did 6 samples (for now) and when I check the quantity/quality of my pre-capture by using the Bioanalyzer 1000 DNA chip I was not totally happy with the results. Effectively, all my 6 curves are more likely with the bad one (I attached an example). So, I understand that I have now several fragments around 300-700 pb. I was wondering about what could be happened? What I did wrong and if I can continue the protocol?

Thank you for your help and any tips will be well received regarding the library preparation

Regards,
David

[nucacidhunter]

It seems that shearing has not resulted in expected fragment length so library size distribution is too large for SureSelect. Average library insert should be around 150 bp.

It is better to prepare another batch of libraries and not to continue with this one. You can check sheared DNA length distribution before proceeding with library prep.

[davalbuq]

Thank you very much for your prompt response. I also thought that the problem could be due during the fragmentation. However, I don't understand why because I did exactly as it says in the protocol..

I will follow your advice and I will not follow the protocol but repeat it and see if this time improves.

[nucacidhunter]

You can check sheared DNA peak size before starting library prep and shear more if it is too large. Peak should be 150-200 bp. Your Covaris could be faulty.

[davalbuq]

I didn't use the Covaris to shear the DNA but an enzymatic reaction (that one evaluated in the Agilent SureSelect kit). For this reason I don't understand where the problem could occurs.

[nucacidhunter]

I wonder which SureSelect kit you have used.

Edit: It seems that you have copied the attached Bianalyzer trace from the SureSelect QXT manual and according to manual it is fine so you can proceed with hybridization and capture. Decreasing input DNA quantity should reduce the average fragment size. Generally larger fragment size will reduce on target reads.

[davalbuq]

I used the SureSelectQXT Target Enrichment for Illumina Multiplexed Sequencing kit. All my samples were around 25 ng/ul using HS Qubit assay. To shear DNA I used the SureSelect QXT Buffer and SureSelect QXT Enzyme Mix ILM as provided in the kit. Maybe I didn't vortex vigorously the samples and reagent mix?

[nucacidhunter]

Larger fragment size indicates higher DNA to transposon ratio which could be result of inaccurate pipette or quantification. It does not seem to be result of insufficient mixing.

Reading between the lines in Agilent manual, it seems that their transposon is inconsistent evidenced by two Bioanalyzer traces that show a large difference in peak size between two preps but according to them it is fine. I would suggest to contact Agilent tech support for an explanation. I think that large peak is non-optimal for human exome capture and will result in reduced on target capture.

[davalbuq]

Thank you very much for your help. After contacting Agilent support it seems that maybe the quantification was not correct. I will try again a new library preparation during this week and see if everything will be better this time..