Problems with adaptor ligation - NEBNExtUltra II

[Jeflo]

Hello to you all,

I want to perform DNA seq and I am having issues in the step of ligating the adaptors to the sequences.

To give you more details: We want to find the different virus detected in an individual. For that purpose, we eliminated the host genome using DNAses and RNAses on the lysate, and then inactivated the nucleases and performed an additional RNA/DNA isolation step with GeneJet Viral DNA/RNA isolation kit. Then, using the Ovation system by Tekan, samples were RT to dsDNA. Then DNA was purified again using Qiagen columns.

At this point, samples were quantified using nanodrop (most of them were at around 100ng/ul with good ratios), and Qubit with equally good results. Size profiles were assessed with Bioanalyzer HS DNA chip, and profiles looked ok.

We performed then library prep with NEBNExtUltra II starting from 300ng and then a qPCR to measure number of sequences containing adaptors, and there was barely any detection. We checked using a Bioanalyzer the profiles again and there was indeed no shift of the samples, what should be observed after the ligation with the adapters.

We repeated the protocol with two samples in parallel, one from our cohort, another a commercial DNA used as a control. The control sample worked perfectly. There was no detection of adapters on our sample.

Has anyone been on a similar situation? Any idea on what may be the issue here?

Thanks,

J

[nucacidhunter]

NEB adapters are short hairpins so you may not see a shift in size, better to check it after PCR. Without PCR step qPCR will not work as they are not full length adapters and lack qPCR primer binding region.

[Jeflo]

Thanks nucacidhunter,
We run a "control" library and we did see a shift on size for this one, although in this case, this was genomic DNA non-derived from virus. Do you think that could be the determining factor?
Thanks again,

J

[nucacidhunter]

I wonder if you could provide the exact name and product # of Ovation kit used. Ovation RNA Amplification System V2 results in single stranded DNA that cannot be used for double stranded adapter ligation. It also primes cDNA synthesis from polyA. If your viral RNA lacks polyA then I wonder where the final product has come from. If you could post Bioanalyzer trace of amplified cDNA and possibly your starting material I can have a look.

[Jeflo]

Many thanks again! The exact kit that used was 7102-08-FG (previously 7102-08) OVATION RNA-SEQ V2 08 .

According to what we found online, this should produce dsDNA molecules that are not dependent of polyA.

Waiting on your answer and many thanks for you help,

J

[nucacidhunter]

Successful ligation with NEB kit requires fragment end-prep (blunting, 5'phosphorylation and 3' adenylation). SPIA uses RNA/DNA hybrid primers that will persist in at least one end of fragments and presence of ribonucleotide will prevent end-prep so they need to be fragmented before end-prep to remove ribonucleotide. If you have used fragmented SPIA amplified cDNA, I would recommend contacting Tecan Techsupport.

[Jeflo]

Will check that out. Many thanks

[praveenhajeri]

How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!


I am trying to use the Ovation RNA seq V2 kit from Nugen or Tecan to amplify low-input RNA (about 1ng).
(https://lifesciences.tecan.com/ovation-low-input-rna-seq-kit-v2?p=tab--1).

The Protocol and workflow of the SPIA amplification process show linear amplification of cDNA, which is single-stranded (As per the schematic provided by them). However, the kit claims it generates double-stranded cDNA, which is not shown in the figure or explained how it is done!!!

Can somebody please help me understand this?

How does the Ovation RNA seq v2 kit generate double-stranded cDNA instead of single-stranded cDNA??!!

Nugen/ tecan suggests using their library preparation kits (for Illumina) which can utilize only dsDNA templates but not single-stranded.
They seem to work well, but I don't understand how the dsDNA is generated in the amplification step (RNA Seq V2 kit).
BMC_SPIA.gif
Please help.