RTSF Genomics Core
http://rtsf.msu.edu/genomics.html
The Agilent BioAnalyzer 2100
Caliper LabChip GX
How It Works
DNA Analysis
RNA Analysis
Low Sample Input
The Ladder
Good RNA
RIN Score: 9.9
Fast Region
Slight Degradation
RIN Score: 8.5
Worse
RIN Score: 6.9
Pretty Bad
RIN Score: 4.6
All The Way There
RIN Score: 3.1
Plant RNA
RIN Score: 7.8
Non-Standard RNAs
Insects, some bacteria, …
RIN Score: N/A
RTSF Genomics Core
http://rtsf.msu.edu/genomics.html
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Library Validation
The Agilent Bioanalyzer (or Caliper GX) is used
to check the quality and size distribution of
your library. This aids in the calculations used
for determining proper loading concentration
for cluster generation.
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DNA 1000 ladder
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RNA Library using the Truseq
mRNA library kit
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Small RNA Library using TruSeq
Protocol
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Sample of a 16 S library
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DNA library using the Illumina
TruSeq protocol
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Illumina DNA TruSeq Nano
Protocol following the 550bp
Range protocol
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Pooled Library of Different Sizes
These can be a little more difficult to get an accurate concentration for
cluster generation because of the varying sizes. Smaller fragments tend to cluster
better than larger fragments.
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Over Amplification
Peaks larger
than 2x your
library size.
Image from Ray Sequerra Illumina presentation
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Sample of SPRI bead carryover
To help avoid this:
• Use a strong magnet
• Allow the supernatant
to clear completely
• Pipette carefully during
the elution step to
avoid disturbing the
bead pellet.
Image from Ray Sequerra Illumina presentation
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Samples of Primer Dimer
Presence of
Peak <125bp
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How Can These Impact Sequencing
This can be a problem
with sequencing
because they can form
clusters . If the % of
primer dimers is high
then you will eat up your
sequence with primer
dimer reads resulting in
less coverage.
Image from Ray Sequerra Illumina presentation
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What Causes Primer Dimers
• Error when doing your gel cut.
• Improper ratio of Ampure beads used.
• Inefficiency of the ligation reaction.
• too little input DNA
• too much input DNA
• Dimers less <100bp cannot be sequenced but
can bind to the flowcell
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What can you do to get the best
library possible?
• Use the recommended input amounts suggested by
the protocol.
• Obtain an accurate concentration: Fluoremetric
reading with Qubit, Picogreen or Ribogreen is
suggested.
• Assess the sample quality
DNA: 260/280 ratio of 1.8 and gel image
RNA: RIN score ≥8 for Bioanalyzer.
• Follow the protocol and best practice procedures as
suggested. If you have questions contact the
Company.
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