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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 16 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. 17 DNA 1000 ladder 18 RNA Library using the Truseq mRNA library kit 19 Small RNA Library using TruSeq Protocol 20 Sample of a 16 S library 21 DNA library using the Illumina TruSeq protocol 22 Illumina DNA TruSeq Nano Protocol following the 550bp Range protocol 23 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. 24 Over Amplification Peaks larger than 2x your library size. Image from Ray Sequerra Illumina presentation 25 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 26 Samples of Primer Dimer Presence of Peak <125bp 27 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 28 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 29 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. 30 31