Resources

Having trouble interpreting your TapeStation results? Want more information on library preparation and Illumina sequencing by synthesis? Check out the resources below.

TapeStation Troubleshooting

 

If you didn't obtain clear data from your TapeStation assay, it is best to determine the source of error before running more samples. Below are some common issues that may affect results. Expand each section to see possible solutions.

 

Description: Samples have not reached the lower end of the gel lane.

Likely cause: High salt concentration in sample.

  1. Solution

    High salt concentration can cause short running within the gel lane, and result in incorrect identification of lower marker peaks in the TapeStation Analysis Software. Please refer to the salt tolerance guidelines for the appropriate assay:

  2. Salt Tolerance Guidelines

    D1000: 20 mM KCl, 60 mM phosphate buffer, 60 mM guanidine-HCl, 240 mM NaCl, 60 mM acetate

    High Sensitivity D1000: 7 mM KCl, 20 mM phosphate buffer, 20 mM guanidine-HCl, 80 mM NaCl, 20 mM acetate

    D5000: 250 mM KCl, 25 mM guanidine-HCL, 125 mM NaCl, 50 mM acetate, 250 mM Tris-HCl, 25 mM MgCl2, 25 mM BSA

    Genomic DNA: N/A

    RNA: 200 mM Tris, 20 mM EDTA, 50 mM NaCl

    High Sensitivity RNA: 10 mM Tris, 1 mM EDTA

Description: Gel image appears squished or stretched in comparison to other lanes.

Likely cause: Incorrect marker peaks detected by TapeStation Analysis Software, or the sample has run concurrently with marker(s).

  1. Solution

    Ensure the correct marker peaks are detected by the TapeStation Analysis Software. Click the “Electropherogram” button and select the appropriate gel lane, then right-click on the electropherogram and select “Add Peak” to add a new peak. Right click on the new peak and select “Assign as Upper or Lower Marker,” whichever is appropriate. Ensure that all the sample peaks are within the recommended sizing range for the application:

  2. Recommended Sizing Ranges

    D1000

    35 - 1000 bp

    High Sensitivity D1000      

    35 - 1000 bp

    D5000

    100 - 5000 bp

    Genomic DNA

    200 - 60,000 bp

    RNA

    100 - 6000 nt

    High Sensitivity RNA

    100 - 6000 nt

Description: In DNA assays, concentration values are calculated using the area of the sample peak compared to the known concentration of the upper marker. In RNA and genomic DNA assays, quantification is calculated using the lower marker.

Likely cause: The TapeStation Analysis Software automatically calls regions of best fit, but sometimes it is incorrect. The user must ensure the sample peaks are properly integrated by manually adjusting the peak when necessary.

  1. Solution

    To select the area under a peak to be integrated, click on the “Region” button in the TapeStation Analysis Software. Select the appropriate gel lane, then right-click on the electropherogram and select “Add Region” to add a new region. Click and drag the edges of the new region to envelope your desired peak. Multiple regions of varying sizes can be added this way.

Description: The marker peaks are not visible due to sample peaks interfering with the signal.

Likely cause: Some sample has run concurrently with marker(s).

  1. Solution

    Ensure that your sample is free of contaminating magnetic beads if you used them in sample clean-up. Ensure your sample is within the recommended sizing range:

  2. Recommended Sizing Ranges

    D1000

    35 - 1000 bp

    High Sensitivity D1000      

    35 - 1000 bp

    D5000

    100 - 5000 bp

    Genomic DNA

    200 - 60,000 bp

    RNA

    100 - 6000 nt

    High Sensitivity RNA

    100 - 6000 nt

Description: Clear peaks for the lower and/or upper marker are visible, but no peaks are present for the sample.

Likely cause: The sample is too dilute or sample size is outside the assay’s sizing range.

  1. Solution

    Concentrate your sample until it is within the recommended range. Ensure your sample size is within the assay’s sizing range:

  2. Accepted Concentration Ranges

    D1000

    0.1 - 50 ng/μL

    High Sensitivity D1000      

    0.1 - 1 ng/μL

    D5000

    0.1 - 50 ng/μL

    Genomic DNA

    10 - 100 ng/μL

    RNA

    25 - 500 ng/μL

    High Sensitivity RNA

    0.5 - 10 ng/μL

     

    Note: you may need to use a High Sensitivity assay if your sample is too dilute.

  3. Recommended Sizing Ranges

    D1000

    35 - 1000 bp

    High Sensitivity D1000      

    35 - 1000 bp

    D5000

    100 - 5000 bp

    Genomic DNA

    200 - 60,000 bp

    RNA

    100 - 6000 nt

    High Sensitivity RNA

    100 - 6000 nt

Description: Bands appear above the upper marker in the gel image.

Likely cause: The sample may be too concentrated for the application.

  1. Solution

    Dilute or concentrate your sample until it is within the recommended range:

  2. Accepted Concentration Ranges

    D1000

    0.1 - 50 ng/μL

    High Sensitivity D1000      

    0.1 - 1 ng/μL

    D5000

    0.1 - 50 ng/μL

    Genomic DNA

    10 - 100 ng/μL

    RNA

    25 - 500 ng/μL

    High Sensitivity RNA

    0.5 - 10 ng/μL

     

    Note: you may need to use a High Sensitivity assay if your sample is too dilute.

Description: Samples which are contaminated with genomic DNA contain a third peak which migrates in the region of the 18 and 28S rRNA. Occasionally this can be mistaken for the 18 or 28S peak.

Likely cause: Genomic DNA is present in the sample.

  1. Solution

    Treat samples with DNAse to eliminate the genomic DNA peak.

Description: The RNA Integrity Value, or RIN, is a measurement of the quality RNA. Total RNA is required for accurate RIN measurement, as it is calculated using the RNA from large and small ribosomal subunits. RIN value is unable to be calculated correctly by TapeStation Analysis Software.

Likely cause: Extremely degraded sample, or sample concentration outside recommended range.

  1. Solution

    Occasionally the TapeStation Analysis Software is unable to detect or call ribosomal RNA peaks in highly-degraded samples.

    For samples outside the assay’s concentration range, dilute or concentrate your sample until it is within the recommended range:

  2. Accepted Concentration Ranges

    RNA

    25 - 500 ng/μL

    High Sensitivity RNA   

    0.5 - 10 ng/μL

     

    Note: you may need to use a High Sensitivity assay if your sample is too dilute.

  1. Message: Caution! Expired ScreenTape

    Description: The ScreenTape was used after its expiration date.

    Solution: ScreenTapes can still function well after expiry, as expiration does not always affect results. No action is required.

  2. Message: Sample concentration outside recommended range/functional range for DIN

    Explanation: The concentration of the sample is out of the concentration specified for the assay.

    Solution: Dilute or concentrate your sample until it is within the recommended range for the application:

    Accepted Concentration Ranges

    D1000: 0.1 - 50 ng/μL

    High Sensitivity D1000: 0.1 - 1 ng/μL

    D5000: 0.1 - 50 ng/μL

    Genomic DNA: 10 - 100 ng/μL

    RNA: 25 - 500 ng/μL

    High Sensitivity RNA: 0.5 - 10 ng/μL

    Note: you may need to use a High Sensitivity assay if your sample is too dilute.

  3. Message: Marker(s) not detected

    Description: The lower and/or upper marker has not been identified by the software. The image is unaligned and no sizing information provided.

    Solution: Manually assign the upper and/or lower marker in the TapeStation Analysis Software. Click the “Electropherogram” button and select the appropriate gel lane, then right-click on the electropherogram and select “Add Peak” to add a new peak. Right click on the new peak and select “Assign as Upper or Lower Marker,” whichever is appropriate.

  4. Message: DIN/RIN edited (Ladder sizing or Marker position changed)

    Description: The lab technician has manually changed the position a ladder or marker peak, and the DIN or RIN may have changed as a result.

    Solution: No action is required.

Additional Bioinformatics Support

 

We are fortunate to have many experts in bioinformatics at the University of Calgary as the importance of big data grows. Below are additional resources outside the CHGI for bioinformatics analysis and assistance.

 

Arnie Charbonneau Cancer Institute

Qingrun Zhang

qingrun.zhang@ucalgary.ca

(403) 220-2833

International Microbiome Centre

Alana Schick

a.schick@ucalgary.ca

 

Hena Ramay

hena.ramay@ucalgary.ca

(403) 210-7770

Energy Bioengineering and Geomicrobiology Group

Xiaoli Dong

xdong@ucalgary.ca

(403) 210-8727


Library Preparation

 

Library preparation is a necessary component in preparing your sample for sequencing. The video below outlines the general steps required to create libraries from input material.

 

Thermo Fisher Scientific

At the CHGI, we commonly use acoustic shearing by Covaris as well as enzymatic fragmentation to create fragments, while PCR amplification is more often used in the creation of 16S libraries.

Adapter sequences are unique for each library, allowing multiple libraries to be pooled in a single multiplex for sequencing on one run.

Due to the high throughput nature of our work, we have incorporated bead-based size selection into our workflows more often than gel electrophoresis.

We use both a TapeStation and qPCR to QC final libraries before loading them on a sequencing run, in line with recommendations from Illumina.


Illumina Sequencing by Synthesis

Illumina

 

 

Once the libraries have been prepared and checked for quality control, equimolar aliquots are pooled to create a multiplex, which is then loaded onto a sequencer to be sequenced.

 

Clusters are generated on a sequencing flow cell as the libraries containing adapter sequences hybridize to a lawn of probes. Illumina technology uses sequencing by synthesis to sequence the clusters in parallel. Fluorescently-labeled nucleotides are enzymatically incorporated one-by-one to the growing chain, and base calls are made from signal intensity measurements after each nucleotide is incorporated.

 

See the video for a general overview, or click here for more details.


Resources for Educators

Illumina's on a mission to improve human health around the world by unlocking the power of the genome. The resources on this website will help you bring genomics into your classroom in dynamic and exciting ways to inspire the next generation—whether you’re an educator or a learner.

The Illumina Foundation and Discovery Education partnered to create DNA Decoded to inspire middle and high school teachers to unlock the power of genomics and impact the future of their students. DNA Decoded provides ready-to-go, standards-aligned lessons and activities for teachers and students to explore the ways they can see genomics in their everyday lives.