RNASeq FAQs

The following set of questions will help you navigate our RNA-Seq service. This may be useful if you are looking to migrate from microarray based gene expression to RNA-Sequencing.

What is RNA-Seq?

How is RNA-Seq different from microarray technology? 

Single reads or paired end reads?

How many reads do I need for my experiment? 

How much sequencing data is guaranteed?

How many samples can I multiplex in a lane?

How much RNA do you need?

What if I have less starting material than indicated in the RNA-Seq Sample Submission Guidelines?

Can I send pre-made RNA-Seq libraries?

What method of RNA enrichment is used by AGRF?

How do I submit my RNA for sequencing?

What data would I get?

How can I access my data and data analysis results?

Will my RNA-Seq data be private and confidential?

 

What is RNA-Seq? 

RNA-Seq is a massively parallel sequencing method for transcriptome analyses. Complementary DNA (cDNA) generated from RNA are sequenced using next-generation sequencing to providing a quantitative view of the RNA present within a sample.

How is RNA-Seq different from microarray technology? 

Unlike microarray, RNA-Seq does not use a pre-defined set of probes to capture and quantify RNA sequences within a sample. Therefore RNA-Seq can be used to investigate both known and novel transcripts.  A further advantage of RNA-Seq is a large dynamic range of expression levels.

Single reads or paired end reads? 

Single read sequencing is efficient for counting transcripts for expression analysis in model organisms.  Paired end sequencing will provide additional coverage of transcripts which is useful for determining the structure of transcripts (i.e. splicing variance) and de novo transcriptome assembly.

How many reads do I need for my experiment? 

The depth of sequencing required for RNA-Seq will depend on the experimental objectives of your project. 10-20 million reads is generally accepted a sufficient for expression analysis, however deeper sequencing may allow detection of rarer transcripts.  We use the following guidelines for determining sequencing depth for RNA-Seq

 

Application 

Read Type 

Clusters (reads) / Sample 

Expression profiling

50 or 100 bp single reads

10-20 million

Alternative splicing

100 bp paired end reads

50-100 million

Allele Specific Expression

100 bp paired end reads

50-100 million

Transcriptome Assembly

100 bp paired end reads

100 + million

How much sequencing data is guaranteed? 

As a guideline, the delivery guarantee is 200million clusters per lane on the Illumina HiSeq 2500. The range is 200-250million clusters on average.

How many samples can I multiplex in a lane? 

Up to 96 with libraries made by AGRF, however up to 384 is possible. The number of samples will depend on the coverage required for your project. Please refer to table 1 above.

How much RNA do you need? 

Refer to sample submission guide

What if I have less starting material than indicated in the RNA-Seq Sample Submission Guidelines? 

We can process as little as 100ng of total RNA for sample RNA-Seq library preparation. It is important to note that our validations are performed with reference RNA material derived from multiple cell lines with a very broad/complex representation of transcripts.  It may not be appropriate to go as low as 100ng for other sources of RNA, therefore we have set a standard lower input of 250ng.  While it is possible to process samples with less than this amount,  sequencing results cannot guaranteed.

Can I send pre-made RNA-Seq libraries ? 

Yes, we can sequence your pre-made RNA-Seq libraries.  You can submit libraries as a pool, or individually and we will perform QC and normalisation on them in preparation for sequencing.  Please refer to the sample submission guidelines for further details.

What method of RNA enrichment is used by AGRF? 

AGRF offers both poly(A) selection (for mRNA) and rRNA depletion to enrich RNA for sample preparation.   mRNA-Seq with poly(A) selection enriches for mature mRNA transcripts and is the most efficient method for quantification of gene expression.  RNA-Seq with rRNA depletion (whole transcriptome sequencing) retains non-rRNA species including pre-mature mRNA and long non-coding RNA.  rRNA depletion is only available using certain species specific kits which inlcude human, mouse, bacterial and plant.

How do I submit my RNA for sequencing? 

Please refer to sample submission guide

 

RNA samples should be sent (on dry-ice) to AGRF’s Melbourne lab along with the sample receipt to the following address:

Melbourne Laboratory

Next Generation Sequencing

Australian Genome Research Facility Ltd.

The Walter and Eliza Hall Institute

1G Royal Pde.

Parkville VIC 3050

 

Before you submit the samples, please contact us so that we can activate your agreement and send login information for your account.

What data would I get? 

Refer RNA-seq-output-formats.pdf

How can I access my data and data analysis results? 

When your sequencing project is complete you will benotified by email.  The data will be available by ftp (login details provided).  Data can be sent on portable media (HDD or USB Flash Drive) for an additional fee.

Will my RNA-Seq data be private and confidential? 

Client confidentiality and protection of Intellectual Property (IP) is of the utmost importance at AGRF.  Ownership of the Results of the Service and Intellectual Property therein will rest in the Clients. You can be confident in the security and privacy of all projects completed with us. 

News

Aboriginal mitogenome analysis shows 50,000 years of connection to country

16 March 2017

Genomic analysis of hair samples collected from Australian Aboriginals across the nation have indicated that cultural connection to country has existed for as many as 50,000 years. 

The Aboriginal Heritage Project, led by the University of Adelaide’s Australian Centre for Ancient DNA recently published their findings in the journal Nature (online 8th March 2017).  AGRF is proud to be associated with the work as part of an ARC linkage grant, with the AGRF’s Operations Manager Dr John Stephen as a Partner Investigator.

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