RNA-seq is the premier method for the analysis of transcript structure and transcript abundance. VANTAGE uses state-of-the-art technology and methodology to produce high quality RNA-seq (transcriptome) data sets. The investigator submits total RNA. VANTAGE will QC the RNA, perform mRNA enrichment and cDNA library preparation. Sequencing is conducted on the Illumina HiSeq 2500. For standard analysis of polyadenylated transcripts VANTAGE employs the Illumina Tru-seq RNA sample prep kit for library preparations, and alternative kits and methods are available for challenging samples such as degraded or low quantity RNA. All samples are indexed and multiplex sequencing is conducted on the HiSeq to generate a dataset with the customer-specified sequence yield and read length in either single or paired end format. Customers are provided with statistics describing the sequencing performance of each multiplex group as well as a demultiplexed FASTQ data file for each sample.
Each sample for RNA-seq is submitted to VANTAGE as total RNA in buffer, at the concentration and volume specified in Table 1. Library preparation is compatible with most buffers containing less than 0.1 μM EDTA, including the elution buffers from standard RNA isolation kits. All samples undergo RNA QC to determine the concentration and to ensure the absence of degradation. Libraries prepared from FFPE or degraded RNA require that DNAse treatment be conducted as a part of the RNA isolation process.
Table 1: RNA-seq Sample Requirements
|Library Preparation Method||Minimum input quantity (ng)||Maximum input quantity (ng)||Minimum conc. (ng/μl)||Maximum input volume (μl)||DNase treatment|
|NuGEN Ovation RNA-seq||1||100||0.2||5||Required|
|NuGEN Ovation FFPE RNA-seq||100||200||20||5||Required|
|Clontech SMARTer low input RNA||0.1||10||0.1||1||Advised|
|Agilent SureSelect Kinome RNA||900||9000||50||18||Advised|
Within each RNA-seq project it is advisable to standardize the quantity of input RNA across all samples. The highest likelihood of success can be obtained by submitting 200 ng of high quality total RNA at a concentration of 10 ng/μl.
Sequencing Quality Control
The quality and integrity of sequencing results are monitored for each data collection run and for each sample. A positive control is incorporated into every sequencing run and it's performance is tracked by VANTAGE to ensure performance. The sequence yield is evaluated for each sample and supplementary data is collected for any sample that does not meet the customer-specified yield.
Delivery of RNA-seq Results
After data collection and demultiplexing are completed and the results have been checked by VANTAGE staff, the researcher is notified by email. This notification includes directions to download the FASTQ files from the VANTAGE LIMS. Customers may also elect to obtain FASTQ files from VANTAGE on an external hard drive.
Library and Data Retention
Sequencing libraries produced by VANTAGE will be maintained as a frozen stock for a minimum of 3 years. Investigators may purchase additional sequencing from any of their VANTAGE libraries. Primary sequencing data (gzip-compressed FASTQ or BAM format) is maintained on the Vanderbilt Bluearc data depot for 90 days after data release.
Investigators interested in RNA-seq of samples that are degraded, from FFPE tissue, or extremely low quantity are invited to consult with the VANTAGE manager regarding the best library preparation approach for their study. Studies of this type cost more than studies of abundant, high quality RNA due to the increased rate at which non-informative sequences are measured. For this reason we suggest that high quality RNA be obtained whenever possible.