Youseq FAQs

If your questions are not listed below, please contact technical support: technical@stratech.co.uk

Why is qPCR better than standard methods for NGS library quantification?

There are disadvantages to nearly all other methods for library quantification. Typical alternative methods include Qubit® (spectrophotometry) or Bioanalyser®/Tapestation® (electrophoresis). Neither of these methods are for suited to high-throughput of samples, requiring laborious and error-prone manual liquid handling. Furthermore they both measure total nucleic acid concentration which is not an accurate measure of sequence-ready library. Our qPCR kit is highly specific in identifying sequence-ready library only. In addition both of these methods lack sensitivity compared to qPCR and are typically more expensive.

What causes variability in qPCR Library quantitation data?

Inaccurate liquid handling is the most common reason for variability. We are happy to give guidance on good pipetting technique and good laboratory practice. Even simple things like ensuring that all standards are thoroughly vortexed to ensure they are correctly resuspended will help to ensure good data quality.

What are the storage recommendations for YouSeq NGS Library Quantification Kits?

Upon receipt, store the kit at -20°C. When stored and handled correctly, the kit components will remain perfect for at least six months from the date of receipt. All components of the kit – are stable for more than 20 freeze/thaw cycles.

Why does the NGS Library QC only have a 4 point standard curve?

Using 4 points instead of (for example) 6 saves previous wells on every plate that you run. The primers in the kit are highly efficient ~100%. We have demonstrated (during our kit development process) that the kit works over a wide dynamic range. Therefore, it is fair to extrapolate the 4 point standard curve in both directions and still deliver highly accurate results for samples that may fall out side of the range of the standard curve.

What is the difference between COVID19 and SARS-CoV-2?

SARS-CoV-2 is the official name of the novel corona virus which originated in 2019 in Wuhan, China. COVID19 is the name given to the disease, to which SARS-CoV-2 causes.

Is this an antigen or antibody test?

Simply, No. These kits detects viral RNA. Meaning the test can detect if the virus is currently present and at what level. It can not detect an immune response.

Will these tests know if I have ever been infected with SARS-CoV-2?

No, this will simply detect whether a sample has an infection or not, when collected.

What does a 'library' mean?

A collection of DNA or cDNA fragments prepared for sequencing by a performing a series of enzymatic steps. These steps are commonly referred to as the Library Preparation.

What are the three types of Reads that can be performed with Illumina based sequencing?

– Single-End Read (SE), which provides sequence from only one end of the DNA insert – Paired-End Read (PE), which provides sequence from both ends of a DNA insert – Mate Pair Read (ME), Similar to paired-end but both reads come from a single strand of DNA

What is read depth?

The number of times a nucleotide is sequenced. The deeper the read depth, the higher degrees of confidence in the base calls.

Can I use your NGS MasterMix with my own home-brew panel?

Yes you can. The MasterMix is designed to be robust and perform well with any good quality NGS panels.

Can I use your custom panel on an iSeq/MiSeq/HiSeq/NovaSeq?

Yes, YouSeq custom panels can be used on all illumina platforms.

How is your custom panel different to other custom panel offerings in the market?

Unlike our competitors YouSeq custom panels are developed by our expert scientists and are not algorithm driven. All our panels are optimised for guaranteed performance delivering high quality sequencing data. YouSeq custom panels also come with all the reagents you need to prepare your library, it’s a complete solution.

What is the minimum sample DNA that I can use?

Due to the targeting regions being amplified using PCR, YouSeq’s kits work with as little as 5 ng of purified genomic DNA.

What is PhiX?

PhiX is a ready-made DNA library. It’s useful as a positive control to check all is working well with your sequencing run. It also adds “complexity” which is useful. DNA sequencers don’t like to sequence multiple identical libraries at the same time so adding PhiX mixes things up a bit and helps your sequencer perform better.

What does polymerase Fidelity mean?

The ability to insert the correct base during a PCR cycle. Therefore, having a high fidelity master mix would mean having a low error base incorporation rate and higher quality libraries

What does Read Length mean?

The number of bases sequenced by your sequencer as a continuous length.

What is the difference between COVID19 and SARS-CoV-2?

SARS-CoV-2 is the official name of the novel corona virus which originated in 2019 in Wuhan, China. COVID19 is the name given to the disease, to which SARS-CoV-2 causes.

Is this an antigen or antibody test?

Simply, No. These kits detects viral RNA. Meaning the test can detect if the virus is currently present and at what level. It can not detect an immune response.

Will these tests know if I have ever been infected with SARS-CoV-2?

No, this will simply detect whether a sample has an infection or not, when collected.

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