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NGS questions
What is included in the One 16S kit?
We supply everything that is required for the library preparation side of the workflow. We would expect the customer to have the usual molecular laboratory tools available to them.
What machines does the One 16S kit work on?
The One 16S NGS kit is compatible with all Illumina platforms. We use iSeq and MiSeq instruments in our lab and they work perfectly.
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 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.
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.
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 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.
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 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 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 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 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 does Read Length mean?
The number of bases sequenced by your sequencer as a continuous length.
qPCR questions
What is the detection profile of the assay?
The detection profile of our assays are described in the specificity section of the relevant handbook. If this section does not list any specific species names, it should be assumed that all species in the genus are detected.
Do you offer extraction kits?
We don't supply extraction kits. However, all our qPCR kits will work with any industry leading extraction kit available to them that derives high quality DNA/RNA.
Are your multiplex kits quantitative?
All our multiplex qPCR kits are designed for qualitative detection of the targets.
Is the positive control genomic DNA?
No, the positive controls supplied with our kits are synthetic versions of the amplicons built into a single stranded DNA oligo. They are then carefully calibrated to a known copy number and stabilised.
What is the shelf life of your products?
All YouSeq products have an expiry date of 1 year from the date of shipping when stored at -20C.
What is the difference between your Internal and Endogenous controls?
Our Endogenous control targets a human gene to confirm that a successful sample was taken. Our Internal extraction control is an exogenous target. It is spiked into the lysis buffer before extraction. This enables users to determine the success of the extraction step.
What if the qPCR kit I’m looking for isn’t on your website?
If the kit you are looking for is not currently in our catalogue, we can certainly custom develop this for you. Please get in contact with us to discuss this further.
What machines do your qPCR kits work on?
Our kits are platform agnostic, and we have a generic protocol which will run on all platforms capable of reading the fluorophores included in the kit.