- Before You Start
- Illumina HT DNA sequencing equipment
- cBot Cluster Generation System
- Covaris S220 Focused Acoustic Shearer
- Agilent Bioanalyzer Equipment
- Nanodrop Quantification Equipment
- Speed-Vac Concentrator System
- Array Automation's ArrayPlex - Automated/Robotic Instrumentation
- Affymetrix microarrays equipment
- PacBio RS
- Fluidigm C1
- BioNano Irys
Before You Start
Contact the GHTF to schedule a consultation about experimental design. Below are a number of commonly asked questions to help you begin planning your experiment. Please consult with us to let us know the time frame of your experiment and to give you an idea of our current workload.
Suggestions for making next gen sequencing libraries
GHTF has experience with kits from Epicentre, BiooScientific, Kapa Biosciences, ZymoResearch, NuGen and Illumina. Commercial kits are changing rapidly towards reducing the protocol time and allowing lower inputs of sample. We are available to discuss the protocols that will be optimal for your experimental design and goals.
For whole exome libraries, the Agilent, Roche (Nimblegen) and Illumina TruSeq all work well.
- Can you suggest any good references in preparing for a sequencing or microarray experiment?
- Applications of next-generation sequencing
- RNA-Seq: a revolutionary tool for transcriptomics
- Advancing RNA-Seq analysis
- Next-generation sequencing transforms today's biology
- Next-Generation Sequencing: From Basic Research to Diagnostics
- Sequencing technologies — the next generation
- Characterizing and Measuring Sequence Bias
- Evaluation of next generation sequencing platforms for population targeted sequencing studies
- Expression Profiling - Best Practices for Data Generation and Interpretation in Clinical Trials.
- The Use and Analysis of Microarray Data.
- Multiple-laboratory comparison of microarray platforms
- Independence and reproducibility across microarray platforms
- Standardizing global gene expression analysis between laboratories and across platforms
- What experimental variables may affect my second generation sequencing or microarray experiments?
- Sample collection and nucleic acid isolation. All variables that you do not want to study should remain the same. In other words, samples should be collected at the same time of day, the same amount of time after watering or feeding.
- DNA and RNA concentration and quality should be checked on a reliable spec. We have a NanoDrop Spectrophotometer available to read low volumes. Make sure the amount of starting material is the same for every library or chip in the experiment. We also have a Qubit fluorometer for quantifying low concentrations of nucleic acids. For next generation sequencing library quantification we recommend Q-PCR quantification with the KAPA Library Quant Kit.
- Using different technicians, equipment and a variety of reagent lots may also affect your results.
- How many replicates should I use in my microarry experiment?
To be able to use standard T-tests, you should have at least 3 replicates. ANOVA would need 5 replicates. The more replicates you have the lower the False Discovery Rate (FDR).