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FAQ

Find answers to Frequently Asked Questions

 

Q:  How do I start a sequencing project?

A:  We highly advise scheduling a free consultation with the facility directory, Amber Scott, by emailing Biof-ngs@colorado.edu or by making a reservation in iLab.  If you have already had a consultation, you may log into iLab and make a Service Request for your sequencing project.

 

Q:  What does a sequencing run cost?

A:  Click here to see pricing.  For a more accurate cost estimate for your specific project, please schedule a consultation with Amber through iLab or by emailing Biof-ngs@colorado.edu.

 

Q:  How do I log into iLab?

A:  Go to https://cu.corefacilities.org/account/login and use your CU identikey to log in.  Your PI must also have an account.  If your PI needs an iLab account, please email .

 

Q:  Why can't I schedule a sequencer?

A:  Only trained users are able to schedule sequencers.  You can, however, look at the sequencing calendar to see the schedule by logging into iLab.  Click here for instructions.  Once we have received your samples we will tentatively schedule sequencing for two days later or when the sequencer is next available.  Two days is required for library QC.   

 

Q:  What does library QC entail?

A:  Libraries are first quantified using Qubit which uses a fluorescent dye specific to double stranded DNA.  Subsequently, the library fragment size is analyzed by TapeStation (Bioanalyzer equivalent) to determine the molarity of the sample.  Additionally, adapter dimer or over-amplification which may skew quantification or interfere with sequencing is detected.  Finally, qPCR is conducted using Illumina specific primers to mimic how the sample will perform on the sequencer.  qPCR ensures the adapters were in fact ligated to the fragments and will cluster on the sequencing flow cell.  Adjustments to the concentration loaded on the sequencer are also made based on the qPCR results and how efficiently the fragments are amplifying.  

NOTE:  Despite thorough QC analysis performed on samples the facility cannot guarantee the performance of any given library.  

 

Q:  What is the qPCR:Qubit ratio?

A:  qPCR:Qubit is a ratio between the concentration of the library determined by qPCR and Qubit.  Generally, the ratio is expected to be around 1 though many factors may skew this ratio.  For example, if fragment sizes are small they tend to amplify more efficiently, resulting in a qPCR:Qubit ratio >1.  If the adapters were not ligated very efficiently, some fragments will not amplify and the qPCR:Qubit ratio will be <1.  The ratio can also help determine if adapter dimers will be an issue or if the library is behaving as expected and worth sequencing. 

 

Q:  Why doesn't my sequencing run start the day I drop off my samples?

A:  Generally, at least two days are required for QC to ensure the samples are ready to sequence.  Further measures may be necessary to prepare the samples for sequencing (removal of adapter dimer, size selection, etc) which may require additional time.

 

Q:  What is the best kit for preparing my library?

A:  The library preparation kit you use will depend largely on your sequencing project:  the concentration and quality of the input DNA/RNA and what data you would like to get out of it.  The best course of action is to schedule a consultation with the facility director, Amber Scott by emailing: Biof-ngs@colorado.edu.

 

Q:  How many reads will I get?

A:  Generally, anywhere between 10 million and 400 million reads.  It depends on the sequencing instrument, kit and the quality of the library.  Click for more detailed information on the MiSeq kits and the NextSeq kits.

 

Q:  What is the difference between single read (SR) and paired end (PE)?  Which should I choose?

A:  In single read sequencing, the sample strand is read from one end to the other in a single direction. In contrast, paired-end sequencing starts at one end, progresses for a set length, then reads from the opposite end of the fragment to meet the copies in the middle. Paired-end can resolve ambiguity in a strand whether it is due to repeat sequences, variants and rearrangements, or due to small fragment size. Paired-end sequencing also allows for sample indexing and for more sample to be processed per flow cell. However, paired-end reads are also more time consuming.

Which you should choose depends on your sequencing project.  We recommend scheduling a consultation with Amber through iLab or by emailing Biof-ngs@colorado.edu to determine the specifications for your sequencing project.

 

Q:  How does Illumina sequencing work?

A:  During library construction an Illumina specific adapter is ligated to the ends of each fragment.  The adapters bind to complementary oligos attached to a flow cell.  Subsequently, fluorescently tagged reversible terminators are added by polymerase.  The fluorescence is captured by images, and the cleaved off to allow the addition of the next base in the following cycle. For more detailed information on Illumina sequencing, please visit their website:  https://www.illumina.com


Q:  What is the MinION?  

A:  The MinION is a small device that performs real time DNA and RNA nanopore sequencing. It can run multiple experiments per flow cell, has adjustable read lengths, and plugs into a computer, being much smaller and more portable than any other sequencer. Instead of measuring a fluorescent signal, the MinION measures disruptions in electric current as molecules pass through the nanopores embedded in a polymer plate. It makes a base call based on the different currents each base generates in the plate. While the MinION has the advantages stated above, it does have a high error rate compared to other sequencing platforms.

 


 

 

 

 

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