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.
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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:
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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).
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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:
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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.
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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).
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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:
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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.
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Solution
Concentrate your sample until it is within the recommended range. Ensure your sample size is within the assay’s sizing range:
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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.
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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.
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Solution
Dilute or concentrate your sample until it is within the recommended range:
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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.
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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.
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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:
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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.
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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.
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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.
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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.
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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.
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.