Frequently Asked Questions
What are the advantages of RNAseq over microarrays for expression analysis?
Microarrays measure an average signal from hybridisation events to particular probes whereas RNAseq is a more direct measurement of transcript abundance. Microarray measurements are affected by cross-hybridisation signals from other transcripts and can only measure transcription from the gene probes they contain - RNAseq does not have these limitations. While the per sample cost of RNAseq is slightly higher than the cheapest microarrays, RNAseq derived expression data is more accurate and has a higher dynamic range. RNAseq data can also be analysed for SNV’s, exon usage and the transcribed DNA strand can be determined.
Which sequencer should I use – Miseq, Nextseq or Novaseq?
With up to 25m reads, the Miseq is suited to organisms with smaller genomes (eg. viruses and bacteria) or targeted sequencing of smaller areas in organisms with larger genomes (eg. amplicon sequencing). The Nextseq offers up to 400m reads and the Novaseq from 800m (SP flowcell) up to 10b (S4 flowcell) reads. We can help you make this decision by discussing the merits of each sequencer in relation to the project aims and budget.
How much starting DNA / RNA do I need?
Many NGS library preparation kits require only 50 - 100ng of RNA or DNA, however, small RNA and whole-transcriptome RNA libraries generally require more material. Where possible, it’s preferable to use larger starting amounts which avoids having to use extra cycles of PCR during the library preparation stage. Where the amount is limited, specialised library kits are also available enabling picogram quantities to be used for some applications.
How many samples can I multiplex in one lane or run, ie, how many indices are there?
For most library systems there are generally at least 16 indices available and some, like NexteraXT, have up to 384.
Can you do library preparation for me?
Yes, for the standard RNA/DNA workflows. Other specialised methods may be possible in consultation.
Can you do the tissue/cell processing including the extraction of RNA/DNA?
No the facility does not generally do front-end experimental work or nucleic acid extraction. Assisteance can be provided with single cell 10X Genomics experiments.
How will I receive my data at the completion of the experiment?
Miseq and Nextseq data is acquired in Basespace (Illumina’s cloud storage) and transferred to the user at the completion of the run. Novaseq data is available to internal SAHMRI users on the HPC drive &/or local network. External users will need to provide a server address for secure copying the data to or a portable drive.
What QC do you perform and how I can be assured my data is of high quality?
All sequencing runs will have sample pools spiked with PhiX to assess run quality. Where sample libraries are prepared by the facility, FastQC metrics will also be monitored to ensure the whole sequencing process is successful.
How much will it cost to do my experiment?
There are many combinations of library preps and sequencing strategies so please contact the facility for a competitive quote.