QIAseq 1-Step Amplicon Library kit-reviews?

[Buckethead84]

Hello all,

Has anyone used the QIAseq 1-step amplicon library kit previously and have any critiques of it? It seems to work very fast by incorporating the most of the steps of library preps into single reaction. They published an interesting protocol last year to get highly multiplexed AmpliSeq panels working on an Illumina platform (attached).

Does anyone have any opinions on what the shortfalls of using something like this would be? I guess the 1-step library prep reaction is probably less efficient since all of the reactions are lumped together, but my understanding is that it wouldn't matter because they're amplicons; meaning even if you only get 1% of your input, there are billions of other copies of that same amplicon that are sequence-able?

Also, what do any of you think about trying to get this library prep protocol to work for sheared gDNA amplified from single cells? Technically all of that gDNA is a type of amplicon at this point since it's derived from a single source. It may be a stretch, but I'm interested in hearing what other people think.

Thanks!

[nucacidhunter]

I have not used the kit. It is marketed for multiplex PCR amplicons because most of them use Taq for amplifications that adds non-template dependent 3’ A overhang. So, basically one step does adapter ligation only and so it cannot be used for shotgun DNA libraries that require end repair and A tailing before ligation. Any ligase (kit or non-kitted) can carry out adapter ligation.

[ecSeq Bioinformatics]

Hi everyone,

our genetic testing software 'Seamless NGS' offers an easy-to-use workflow for the analysis of Qiagen's QiaSeq panels.

Before mapping the reads, the common sequence and the UMIs are clipped away. After mapping, the UMIs sequences are properly used to correct for PCR clones and the single amplicon primer is clipped away to ensure correct and unbiased variant calling. The workflow was developed, tested and validated together with a trustworthy pathological laboratory at one of the oldest university hospitals in Germany. Having long-lasting experience in that field, together with them, ecSeq was able to get the most out of the QiaSeq panel.

If you are interested in our solution, please check www.seamless-neg.com and/or write us a short email ([email protected]) to make an appointment for a personal live demo (online).